Store-operated calcium cellular assay

ABSTRACT

The present technology provides a cell based assay for identifying compounds that modulate store-operated ionic calcium levels using itpr mutant cell lines, such as itpr-ku cells, which have abnormal ionic calcium levels.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/628,064, filed Nov. 30, 2009, which is hereby incorporated byreference in its entirety.

FIELD OF THE TECHNOLOGY

The field of the present technology relates to, among others, cell basedassays for compound identification.

BACKGROUND

The major entry pathway of calcium ions (Ca²⁺) in electricallynonexcitable cells is the store-operated calcium channel (SOC). Thestore-operated channel is encoded by the Orai gene and is the poreforming subunit of the Ca²⁺-release activated Ca²⁺ (CRAC) channel. SeePrakriya, M. et al., Nature 443, 230-233 (2006); Vig, M. et al., CurrBiol 16, 2073-2079 (2006); and Yeromin, A. V., et al., Nature 443,226-229 (2006). When intracellular stores of Ca²⁺ are low or depleted,then Ca²⁺ influx through the store-operated calcium channel isactivated.

In the context of neuronal activity, calcium ions act as intracellularmessengers during synaptic transmission and in developmental processes.Specific attributes of a Ca²⁺ “signature” such as, amplitude, durationand frequency of the signal can determine the morphology of a neuralcircuit by affecting the outcome of cell migration, the direction takenby a growth-cone, dendritic development and synaptogenesis. SeeBerridge, M. J., Neuron, 21, 13-26 (1998). Ca²⁺ signals also determinethe nature and strength of neural connections in a circuit by specifyingneurotransmitters and receptors. See Borodinsky, L. N. & Spitzer, N. C.,Proc Natl Acad Sci USA, 104, 335-340 (2007). Thus, neuronal Ca²⁺ signalscan affect excitability and neural circuit formation.

The inositol 1,4,5-trisphosphate receptor (InsP3R, itpr) gene is aligand gated Ca²⁺-channel present on the membranes of intracellular Ca²⁺stores. See Banerjee, S. et al., J Neurosci 24, 7869-7878 (2004); Joshi,R. et al., Genetics 166, 225-236 (2004). It is thought to be involved invarious aspects of neuronal function including excitability,neurotransmitter release, synaptic plasticity and gene transcription.See Banerjee, S. & Hasan, G., Bioessays, 27, 1035-1047 (2005); Berridge,M. J., Neuron 21, 13-26 (1998). Mutants in the gene coding for the mouseInsP3R1 are ataxic. See Matsumoto, M. et al., Nature 379, 168-171(1996); Street, V. A. et al., J Neurosci., 17, 635-645 (1997).

SUMMARY

An aspect of the present technology is a method of identifying acompound that modulates store-operated calcium entry levels in a cell,comprising providing a test compound to an itpr-ku mutant cell; anddetermining whether the test compound increases or decreases theabnormal store-operated calcium entry level of the itpr-ku cell, whereina test compound that increases or decreases the calcium release throughInsP3R and/or changes the store operated calcium entry in an itpr-kumutant cell is a compound that modulates store-operated calcium entrylevels. In one embodiment, the store-operated calcium entry level of theitpr-ku cell either increases or decreases to a level that approximatesthe calcium level of a control cell. In one embodiment control cell is(i) a wild-type, normal cell, (ii) a cell with a dOrai/Kum-170; itpr-kumutant genotype, or (iii) a cell without the itpr-ku mutant genotype.

In a further embodiment, determining whether the test compound modulatesthe abnormal store-operated calcium entry level of the itpr-ku cell isachieved by performing calcium imaging of the store-operated calciumentry environment of the itpr-ku cell, and comparing the calcium imagingpatterns before and after the addition of the test compound to theitpr-ku cell.

In another embodiment, the method further comprises comparing thecalcium imaging patterns of the itpr-ku cell with the calcium imaging ofthe store-operated calcium entry environment of the control cell,wherein a similarity in calcium imaging patterns between the two cellsindicates the test compound can modulate the store-operated calciumentry level of the itpr-ku cell. In one embodiment, the control cell isa (i) a wild-type, normal cell, (ii) a cell with a dOrai/Kum-170;itpr-ku mutant genotype, or (iii) a cell without the itpr-ku mutantgenotype.

In another embodiment, the test compound is selected from the groupconsisting of a small molecule, an inorganic compound, an organiccompound, a biomolecule, a chemical, a protein, a peptide, or a nucleicacid.

A method for treating a disease characterized by abnormal store-operatedionic calcium levels comprising administering a compound identified bythe method of the present technology to an individual with such adisease. In one embodiment, the disease is severe combinedimmunodeficiency, acute pancreatitis, or Alzheimer's Disease.

In one embodiment, therefore, is a method of identifying a therapeuticcompound useful for treating a disease that is characterized by anabnormal intracellular calcium level, comprising: (A) providing acandidate therapeutic compound to a cell which (i) comprises a mutatedinositol 1,4,5-trisphosphate receptor gene and which (ii) ischaracterized by an abnormal intracellular calcium level compared to thecalcium level of an equivalent cell which does not comprise the mutatedinositol 1,4,5-trisphosphate receptor gene; (B) determining whether thecandidate therapeutic compound increases or decreases the abnormalintracellular calcium level of the cell, and, if it does, then, (C)administering the candidate therapeutic compound to cells isolated froman individual who has a disease characterized by abnormal intracellularcalcium levels; and (D) determining (i) whether the candidatetherapeutic compound increases or decreases the abnormal intracellularcalcium level of the isolated cells, and (ii) comparing any increase ordecrease in the abnormal intracellular calcium level against theintracellular calcium level of normal cells isolated from a healthyindividual who does not have the disease; wherein a candidatetherapeutic compound that effectuates a change in the intracellularcalcium level of the diseased cells toward the intracellular calciumlevel of the normal cells, is identified as a therapeutic compounduseful for treating a disease that is characterized by an abnormalintracellular calcium level.

In one embodiment, the cell of (A) expresses no inositol1,4,5-trisphosphate receptor gene other than the mutated inositol1,4,5-trisphosphate receptor gene. In another embodiment, the mutatedinositol 1,4,5-trisphosphate receptor gene comprises the mutationscharacteristic of the itpr-ku mutated gene, or mutations that areequivalent to the itpr-ku mutated gene. In another embodiment, the cellof (A) is a DT40 cell. In another embodiment, the cell is a Drosophilacell and the mutated inositol 1,4,5-trisphosphate receptor gene has thesequence of the itpr-ku mutated gene. In another embodiment, the cell of(A) is a mammalian cell. In a further embodiment, the cell of (A) is ahuman cell, and the isolated cells of the diseased individual of (C) arehuman cells. In one embodiment, the diseased cells are cells isolatedfrom a human who has a disease selected from the group consisting ofspino-cerebellar ataxia; severe combined immuno-deficiency, Darier'sdisease, an immunodeficiency, acute pancreatitis, Alzheimer's Disease.

In another aspect, the method further comprises administering thetherapeutic compound to (i) the same individual from whom the diseasedcells of (C) were isolated, or (ii) an individual who has the samedisease or same type of disease as the individual from whom the diseasedcells of (C) were isolated. In one embodiment, the therapeutic compoundis formulated into a pharmaceutical formulation prior to administrationto the individual. In one embodiment, the individual is a human male orhuman female.

In another aspect, the method comprises (i) imaging the intracellularcalcium levels of cells isolated from the individual before and afteradministration of the therapeutic compound or the pharmaceuticalformulation of the therapeutic compound to the individual, and (ii)determining if the abnormal intracellular calcium levels that arecharacteristic of the disease has changed toward the level ofintracellular calcium levels of a normal, healthy cell. In oneembodiment, the method further comprises administering the same or anincreased or decreased amount of the pharmaceutical formulation to theindividual and re-imaging the intracellular calcium levels of cellsisolated from the individual to determine if the intracellular calciumlevel is near or at or is approximating the intracellular calcium levelof a normal, healthy cell.

The foregoing summary is illustrative only and is not intended to be inany way limiting. In addition to the illustrative aspects, embodiments,and features described above, further aspects, embodiments, and featureswill become apparent by reference to the drawings and the followingdetailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Illustrative embodiments demonstrating that intracellular Ca²⁺homeostasis in larval neurons is altered upon RNAi knock down of dOraiand dSTIM:

(a) Pseudocolour image representation of one embodiment of themeasurement of store Ca²⁺ and store operated Ca²⁺ entry (SOCE) inprimary cultures of neurons loaded with a Ca²⁺ sensitive dye (Fluo-4)from wild type (WT) larvae and those in which either dOrai or dSTIM isknocked down using RNAi. Store Ca²⁺ was measured by depleting storesupon addition of 10 μM thapsigargin (Tpg). SOCE was monitored byinclusion of Ca²⁺ (to a free concentration of 1 mM) at t=225 s, to theextracellular buffer. Scale bar represents 10 μm. Warmer colorsrepresent higher Ca²⁺;

(b) Single cell traces of SOCE by Ca²⁺ add-back after store depletionwith thapsigargin. Arrows represent the peak values of response whichhas been plotted as a box chart in FIG. 1 c;

(c) Box plots of the ΔF/F values of SOCE in neurons where the store hasbeen depleted with thapsigargin treatment followed by addition of 1 mMCaCl₂ to the bath. The bigger boxes represent the spread of the ΔF/Fvalues, the smaller squares represent the mean value and the diamonds oneither side of the boxes represent outlier values. SOCE is severelycompromised in neurons where dOrai (*PANOVA=0.04281) or dSTIM(**PANOVA=0.5875 E-7) transcripts are down regulated compared to WT.Nearly 70-80% of cells that respond to thapsigargin treatment havedetectable SOC and this was comparable between all the genotypes tested;

(d) Box plot comparison of ER store Ca²⁺ levels between neurons of WT,RNAi controls and those with pan-neuronal down-regulation of dOrai ordSTIM. At least 150 cells of each genotype were analyzed to obtain anestimate of [Ca²⁺]ER which is significantly lower in cells where Orai(*PANOVA=2.61 E-6) or STIM (**PANOVA=0.001) is downregulated as comparedto WT;

(e) Kolmogorov-Smirnov (K-S) plot analyzing the distribution ofintracellular Ca²⁺ or [Ca²⁺]i in neurons loaded with the Ca²⁺ sensitiveratiometric dye Indo-1. The frequency distribution is significantlyshifted to the left for cells where dSTIM is knocked down, indicating ahigher frequency of cells with reduced resting Ca²⁺ levels(PK-S=0.01173); and

(f) A box plot depiction of [Ca²⁺]i in 170 or more neuronsdown-regulated for dOrai or dSTIM with appropriate controls. [Ca²⁺]i wasdetermined using the Grynkiewicz equation44. [Ca²⁺]i in neurons withreduced dSTIM is significantly lower than in WT neurons(*PANOVA=0.02092).

FIG. 2 Illustrative embodiments showing that targeted RNAi knock down ofdOrai or dSTIM in subsets of neurons gives rise to flight motor defects:

(a) Pan-neuronal knock-down of dOrai by an inducible RNAi constructinduces a mild change in wing posture not seen when it is knocked downin glutamatergic neurons;

(b) Pan-neuronal down-regulation of dSTIM transcripts induces asignificant defect in the wing posture not observed when thedown-regulation is restricted to glutamatergic neurons;

(c) Targeting dOrai or dSTIM RNAi to all post-mitotic neurons or theglutamatergic subset of neurons introduces significant flight defects(determined using Student's t-test for two populations) as tested by thecylinder drop test. Flight defects observed upon pan neural downregulation of dSTIM are nearly 100% (**P=1.01 E-8) while down regulationof dOrai results in 60% flies being flight defective (**P=1.9 E-4). Downregulation of dOrai or dSTIM in glutamatergic neurons results in aflight defect of nearly 40% (*P=0.00246 for dsdOrai). All GAL4 controlflies exhibited normal flight. Plotted is the data for glutamatergicGAL4 control. Values are plotted as mean±SE;

(d) dOrai or dSTIM RNAi expression by glutamatergic and pan-neuronaldrivers introduces defects in air puff induced flight patterns among thenon-fliers selected from the cylinder drop test. Flies with downregulated dOrai and dSTIM were unable to sustain flight beyond 5 s andexhibit arrhythmic flight patterns. The numbers in brackets representthe number of flies exhibiting the characteristic pattern of electricalactivity upon air puff stimulation out of the total number of non-flierstested. Arrow indicates the point of delivery of air puff stimulus;

(e) Significantly higher levels of spontaneous firing, was recorded fromthe DLMs of flies where dOrai or dSTIM had been knocked down in eitherall post-mitotic neurons (*P=2.93 E-5 for dsdOrai, 0.04354 for dsdSTIM)or only glutamatergic neurons (*P=0.00456 for dsdOrai, 0.543 E-4 fordsdSTIM). The GAL4 control refers to glutamatergic GAL4. The pan-neuralGAL4 control looked similar. The frequency of spontaneous firing forindividual flies in each genotype is also shown (*). Values are plottedas mean±SE and significance is determined using Student's t-test for twopopulations; and

(f) Representative traces of spontaneous firing activity from the DLMsof the indicated genotypes.

FIG. 3 Illustrative embodiment showing that over-expression of dOrai+ inneuronal subsets causes partial suppression of flight defects in an itprmutant:

(a) Over-expression of UASdOrai+ in aminergic and Drosophilainsulin-like peptide 2 (Dilp2) producing neurons in itprku partiallysuppresses the characteristic wing posture defect of itprku;

(b) Air-puff stimulus elicits flight patterns in itprku fliesover-expressing dOrai+ in aminergic, glutamatergic and dILP-2 producingcells; flight patterns are not sustained beyond 5 s. Arrows indicate thepoint of delivery of an air puff stimulus;

(c) Ubiquitous over-expression of dOrai+ (two copies) by a leakyheat-shock (hs) GAL4 at 25° C. or in sub-neuronal domains suppresses thespontaneous firing of itprku flies (**P=0.00082 using hs GAL4, 0.00133in aminergic cells, 5.54 E-6 in Dilp2 cells and 2.02 E-6 when expressedin the glutamatergic domain; significance is determined by the Student'st-test). Histograms represent mean values of spontaneous firingfrequency while the error bars represent SE;

(d) Cell type specific suppression of neural hyperactivity is seen whendOrai+ is over expressed using aminergic, glutamatergic and Dilp2 GAL4sin the genetic background of itprku;

(e) Delivery of a heat-shock to either 24 hr pupae or 1 day old adultsexpressing 2 copies of dOrai+ under the ubiquitous heat-shock promoterin itprku, enables flies to initiate arrhythmic flight patterns inresponse to an air puff stimulus. In the absence of a heat-shock flightinitiation is not observed. Heat shock induced ubiquitous expression ofdOrai+ also results in partial suppression of the wing posture defect initprku; and

(f) Representative traces of flight patterns from DLMs after delivery ofan air puff stimulus (arrow).

FIG. 4 Illustrative embodiments showing dOrai+ over-expression in itprkuneurons restores intracellular Ca²⁺ homeostasis:

(a) Single cell traces of SOCE by Ca²⁺ add-back after store depletionwith thapsigargin (Tpg). Arrows represent the peak values of responsewhich has been plotted as a box chart in FIG. 4 b;

(b) SOCE was measured by Ca²⁺ add-back experiments. In neuronsover-expressing dOrai+ in itprku, SOCE is significantly higher thanitprku (*PANOVA=0.00209 compared to itprku); it is not altered whendOrai+ is expressed on its own. Pan-neural expression was achieved usingElavC155GAL4 strain;

(c) Box plot representing ER store Ca²⁺ levels in neurons of theindicated genotypes. [Ca2+]ER in itprku neurons is significantly greaterthan wild-type (**PANOVA=5.551 E-6). Pan-neuronal over-expression ofdOrai+ restores normal [Ca2+]ER to itprku. Store Ca²⁺ in cellsover-expressing dOrai+ is similar to WT;

(d) K-S plot analyzing the distribution of intracellular Ca²⁺ in neuronsof indicated genotypes. A greater frequency of cells with higher [Ca²⁺]iis seen in genotypes expressing dOrai+ (PK-S=0.0011); and

(e) Box plot analysis of resting cytosolic Ca²⁺ in neurons withover-expression of dOrai+. The average basal Ca²⁺ in cellsover-expressing dOrai+ with or without itprku in the background issignificantly higher (*PANOVA=0.04621 and 0.0095 respectively) than inWT. Mean resting cytosolic Ca²⁺ in neurons of itprku is similar to WT.170 or more cells were analyzed for each genotype in every experiment.

FIG. 5 Illustrative embodiments showing flight and associatedphysiological defects are suppressed in a combination of dOrai, dSERCAand itpr mutants:

(a) Wing posture defect of itprku is partially suppressed in dOrai2/+;itprku and dOrai1/+; itprku flies grown at 25° C. Triple mutants ofKum170/dOrai2; itprku show better suppression of wing posture defectthan the double mutants;

(b) Flight defect of itprku is neither suppressed by dOrai mutants(dOrai1 or dOrai2) nor Kum 170/+(90-95% defect as assayed by thecylinder drop test, *P=0.00529 compared to WT). A double mutant rescueof itprku with Kum170 and dOrai2 or dOrai1 suppresses the flight defectto 60% (**P=4.91 E-6 in presence of dOrai1 and 5.61 E-4 in presence ofdOrai2, compared to itprku). Marginally greater suppression is achievedwith dOrai1 than with dOrai2. Histograms represent the mean±SE;significance was tested by the Students t-test for two populations;

(c) Snapshots taken within first 5 s of air puff induced flightinitiation in 1, itprku; 2, Kum 170/dOrai2; itprku;

(d) Defects in wing-posture induced by pan-neuronal knock down of dSTIMare suppressed differentially by a single copy of dOrai2 or Kum170.While nearly 50% of flies with dOrai2/+ in the background of lowereddSTIM levels exhibit normal wings, suppression by Kum170/+ is observedfor only 10% of the flies;

(e) Air puff stimulus elicits brief (<5 s) rhythmic flight patterns fromthe DLMs of dOrai2/+; itprku and dOrai1/+; itprku similar to Kum170/+;itprku but not from DLMs of itpr mutants alone. The action potentialsgenerated in 9 out of 17 flies tested for dOrai2/+; itprku and 11 out of19 flies tested for dOrai1/+; itprku tested are rhythmic, accompanied bywing beating which terminated within 5 s of initiation. Triple mutantfliers of Kum170/dOrai2; itprku initiate sustainable rhythmic flightpatterns similar to WT which last for a minimum time of 30 seconds.Similar air puff induced flight patterns are seen in recordings fromflies of Kum170/dOrai1; itprku. A single copy of dOrai2 also restoresrhythmic flight initiation in a majority of flies with pan-neuronal RNAiknock-down of dSTIM. Arrows indicate the point of air puff delivery;

(f) Spontaneous hyperactivity in DLMs of itprku is suppressed by thepresence of a single copy of either dOrai1/+ or dOrai2/+ or the combinedpresence of Kum170/dOrai2 (*P=9.26 E-6, 1.03 E-3 and 1.94 E-4respectively, compared to itprku). High spontaneous firing rates inducedby pan-neuronal dsdSTIM expression are also suppressed by dOrai2/+(*P=0.03537). Recordings were obtained from at least 15 flies of everygenotype and the data plotted as mean±SE. Individual data points havebeen included to indicate the spread; and

(g) Representative traces of spontaneous firing activity from the DLMsof the indicated genotypes. A single copy of a dOrai hypermorphic allelecan suppress spontaneous firing DLMs from of itprku and pan-neuronaldsdSTIM (ElavC155GAL4; UASdsdSTIM) adults.

FIG. 6 Illustrative embodiments showing dOrai and dSERCA mutants alterdifferent aspects of intracellular Ca²⁺ release in Drosophila neurons:

(a) Changes in stimulated Ca²⁺ release through the InsP3R (measured asΔF/F) in itprku alone and in double and triple mutants of itprku, Kum170and dOrai2 is shown in response to 10 μM and 20 μM carbachol. Carbacholresponses from all neurons were obtained by pan-neuronal expression ofthe Drosophila muscarinic acetylcholine receptor (ElavC155GAL4;UASdmAchR) and loading with Fluo-4. Release through the InsP3R issignificantly compromised in itprku (**PANOVA=5.76 E-5 compared to WTfor 10 μM carbachol stimulation) and is similar in Kum170/+; itprku(**PANOVA=3.84 E-6 compared to WT) Presence of dOrai2 in itprkubackground restores InsP3 stimulated Ca²⁺ release to WT levels with orwithout Kum170 (*PANOVA=6.56 E-4 compared to itprku). Results plottedhere are observations from 150 or more cells;

(b) Effect of Kum170 and dOrai2 on perdurance of InsP3-mediatedCa²⁺-release signals. Presence of a single copy of Kum170 alone or indouble or triple mutant combination with dOrai2 or itprku significantlydelays the rate of cytoplasmic Ca²⁺ clearance after carbachol stimulatedCa²⁺-release. Approximately 40-50 cells of each genotype, with similarpeak response times were selected for this analysis. Ca²⁺ clearance timein itpr mutants alone or in combination with dOrai mutants is similar toWT.

(c) Ca²⁺ release through InsP3R is significantly compromised in itprkuand is restored back in double mutants of itprku and dOrai2. Images werepseudo-colored to represent [Ca²⁺]i. Larval neurons from WT, itprku anddOrai2; itprku expressing mAChR were stimulated with 10 μM carbachol andfluorescent images were taken in the time lapse mode. The arrowrepresents addition of 10 μM carbachol. The scale bar represents 10 μm.Warmer colors represent higher Ca²⁺;

(d) Single cell traces of SOCE by Ca²⁺ add back after store depletion bythapsigargin;

(e) SOCE was measured by Ca²⁺ add-back experiments in cells derived fromlarval brains of Kum170 and dOrai2 in the genetic background of itprku.SOCE in neurons derived from Kum170; itprku brains remains low(**PANOVA=2.04 E-4 compared to WT) reminiscent of itprku and unlikecells isolated from Kum170/+ organisms, where it is significantly higherthan WT (*PANOVA=8.11 E-6). The proportion of cells with detectable SOCEin neurons from Kum170; itprku brains is 70%. SOCE remains low indOrai2/+; itprku, though the proportion of cells with detectable SOCE inneurons of this genotype is restored to 65%. SOCE is restored in triplemutants of Kum170/dOrai2; itprku (**PANOVA=0.00667 compared to itprku).Heterozygous dOrai2/+ partially restores SOCE in neurons in which dSTIMis down-regulated by RNAi (**PANOVA=0.00324 compared to pan-neuronaldsdSTIM); and

(f) Box plots representing [Ca²⁺]ER levels of neuronal cells of theindicated genotypes. Double mutants of Kum170 and itprku havesignificantly reduced levels of store Ca²⁺ (**PANOVA=0.00011 compared toWT); dOrai2/+ restores store Ca²⁺ levels in double mutants (dOrai2/+;itprku, *PANOVA=4.73 E-6 compared to itprku) but not in triple mutants(dOrai2/Kum170; itprku; PANOVA=0.03204 compared to WT). Presence of asingle copy of dOrai2 restores [Ca²⁺] ER in neurons in which dSTIM istranscriptionaly down-regulated by pan-neuronal expression of dsdSTIM(**PANOVA=3.34 E-6) compared to pan-neuronal dsdSTIM alone).

FIG. 7 Model of InsP3-mediated intracellular Ca²⁺ signals in itprkumutant neurons and their modulation by Orai and SERCA in the context ofDrosophila flight Illustrative embodiments of the model showing changesin cytosolic Ca²⁺ generated by stimulating hypomorphic InsP3Rs insingle, double and triple mutant conditions. Warmer colors denote higherconcentration of Ca²⁺ in the ER store or cytoplasm. Panel (a) Changes toa single response peak; the black dotted trace represents itprku andother dotted traces represent the indicated double and triple mutantgenotypes; response of the wild type InsP3R is represented by a solidbrown trace. Panel (b) Changes over longer times showing the amplitudeand duration of Ca²⁺ transients upon repeated stimulations of the InsP3Rwhich would be subject to ER store refilling by SOCE. The recurring Ca²⁺wave shown in (b) has not been demonstrated experimentally in ourconditions.

FIG. 8 Illustrative embodiments showing targeted down regulation ofdOrai and dSTIM in neurons:

(a) Targeted expression of UASdOraiRNAi^(221/+) to post-mitotic neuronsresults in a consistently reduction in the level of dOrai transcripts.RT-PCR for dOrai (173 bp) was done on total RNA isolated from 24 hrpupae and normalized to levels of rp49 transcripts (120 bp). Lanes 1 and2 contain PCR products for dOrai and lanes 1′ and 2′ contain PCRproducts for rp49. Lanes 1, 1′ are UASdOraiRNAi^(221/+), lanes 2, 2′ areElav^(C155)GAL4; UASdOraiRNAi²²¹.

(b) Quantification of dOrai transcript levels normalized to rp49transcript levels calculated from RT-PCR products obtained from threedifferent batches of RNA. The ratio of fluorescence intensity of eachPCR product was estimated using Image J software (NIH, USA). Theintensity of the dOrai band was normalized to that of the rp49 band.dOrai transcript levels in UASdOraiRNAi^(221/+) (1.63±0.103) weresignificantly higher as compared to Elav^(C155)GAL4; UASdOraiRNAi²²¹(1.25±0.073) (*P=0.03978 as determined by the Student's t-test);

(c) Targeted expression of UASdSTIMRNAf^(073/+) to post-mitotic neuronsresults in a significant reduction in dSTIM transcript levels (312 bp)as assayed by RT-PCR. Total RNA was isolated from 3^(rd) instar larvalbrains of UASdSTIMRNAI-^(073/+) (1, 1′) and Elav^(C155)GAL4;UASdSTIMiRNAi⁰⁷³ (2, 2′) organisms;

(d) Down-regulation of dSTIM transcript levels by RNAi was determined bymeasuring the ratio of fluorescence intensity of the dSTIM products tothe rp49 products. The ratio measures 0.54±0.05 as compared to 0.79±0.04of controls (*P=0.01725 calculated by the Student's t-test);

(e) Down-regulation of transcripts of dNR1 in RNA extracted from 3^(rd)instar larvae ubiquitously expressing UASdNR1RNAi^(333/+) under aheat-shock promoter. Total RNA was isolated 24 hrs post heat shock, andRT-PCR carried out for dNR1 (154 bp) and rp49 mRNA. Lanes 1, 1′ areheterozygous controls with UASdNR1RNAi^(333/+), lanes 2, 2′ are hsGAL4;UASdNR1RNAi³³³; and

(f) [Ca²⁺]_(ER) in neurons from larvae with pan neural expression ofdsdNR1 is similar to that in neurons of heterozygous dsdNR1 controls.Pan-neuronal knockdown of dNR1 does not alter the SOCE in neuronsfollowing store depletion.

FIG. 9 Illustrative embodiments of characteristics of intracellular Ca²⁺signals in itpr^(ku) grown at 18° C.:

(a) Ca²⁺ add back experiments were done to measure SOC influx followingstore depletion in neurons of WT and itpr^(ku) derived from larvae grownat 18° C. Box plots represent the spread of SOCE between the twopopulations which appear similar. 150 or more cells were analyzed forall the genotypes for each experiment;

(b) Store depletion was done with 10 μM thapsigargin to assay levels ofstore Ca²⁺ in neurons derived from larvae reared in cold. Box plotsrepresenting the range of store Ca²⁺ levels between the two populationsshow that unlike at 25° C., store Ca²⁺ in cells of itpr^(ku) and WTgrown at 18° C. is similar (P_(ANOVA)=0.44044); and

(c) Transcript levels of mAChR were assayed by RT-PCR in total RNAderived from 3^(rd) instar larval brains (mAChR expression wasspecifically targeted to larval neurons) and normalized to rp49. Lanes1-3 contain PCR products for mAChR (463 bp); lanes 1′-3′ contain PCRproducts for rp49. Lanes 1, 1′ control 1, lanes 2, 2′ itpr^(ku) andlanes 3, 3′ itpr^(k/+). The mAChR transcript levels appear identical inall the genotypes tested; and

(d) Box plot of the ΔF/F values in neurons from WT and itpr^(ku) inresponse to stimulation with 5, 10, 20 and 40 μM of carbachol. Ca²⁺release through InsP₃R in response to carbachol is significantlyattenuated in itpr^(ku) upon stimulation with 10 μM, 20 μM and 40 μMcarbachol (**P_(ANOVA)=5.76 E-5, 0.00393 and 1.55 E-6 for 10, 20 and 40μM carbachol stimulation respectively; P-values are all compared to WT).170 or more cells were analyzed per genotype for each concentration ofcarbachol.

FIG. 10 Identification of a P-element insertion dOrai gene in dOrai¹ anddOrai². Position of the EP{gy2}insert in dOrai¹ and dOrai² was obtainedfrom thewww.ncbi.nim.nih.gov/entrez/viewer.fcgi?db=nucleotide&tool=FlyBase&val=CL705882 database. For confirmation the region was amplified from genomic DNAisolated from the two strains. The forward primer used for amplificationmaps to the 5′ end of the Orai gene and the reverse primer iscomplementary to the P-element end. The amplicons were sequenced tore-ascertain the positions of the P-elements in the two strains(arrows).

FIG. 11 Illustrative embodiments showing dOrai¹ and dOrai² suppresslarval lethality of itpr^(ku) at 18° and elevate resting cytosolic Ca²⁺:

(a) Larval lethality in itpr^(ku) is partially suppressed by dOrai^(1/+)and dOrai^(2/+). Error bars indicate S.E. (**P=0.00212 compared toitpr^(ku));

(b) Box plot representation of [Ca²⁺]_(ER) and SOCE in the larvalneurons of dOrai mutants. Intracellular store Ca²⁺ and SOCE in neuronsof dOrai^(1/+) and dOrai^(2/+) are similar to WT;

(c) K-S plot analyzing the distribution of intracellular Ca²⁺ in neuronsof indicated genotypes. The distribution is shifted towards the right inheterozygotes of Kum¹⁷⁰ or dOrai² with or without itpr^(ku), indicatinga higher frequency of cells with elevated [Ca²⁺]i (P_(K-S)=0.001); and

(d) Box plot analysis of resting cytosolic Ca²⁺ in neurons of itpr^(ku)with Kum^(170/+) or dOrai^(2/+) or both. The average basal Ca²⁺ in cellsharboring either dOrai² or Kum¹⁷⁰ with or without itpr^(ku) in thebackground is significantly higher than WT (**P_(ANOVA)=0.0395 fordOrai^(2/+) and 0.0089 for Kum^(170/+)). 170 or more cells were analyzedfor each genotype in every experiment.

FIG. 12 Calcium imaging assay.

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying drawings, which form a part hereof. In the drawings,similar symbols typically identify similar components, unless contextdictates otherwise. The illustrative embodiments described in thedetailed description, drawings, and claims are not meant to be limiting.Other embodiments may be utilized, and other changes may be made,without departing from the spirit or scope of the subject matterpresented here.

The present technology provides a cell-based assay for identifying acompound that modulates store-operated, intracellular calcium levels ina cell that expresses a mutated inositol 1,4,5-trisphosphate receptor(itpr) gene. An itpr cell mutant cell of the present technology hasabnormal levels of store operated, intracellular ionic calcium. InDrosophila, this abnormal level of store operated, intracellular calciumcan be restored by the expression of certain other genes, such as bydOrai (see text and Examples below). In human cells, the itpr gene has ahomolog known as InsP3R, and three ORAI gene isoforms, ORAI1 (FLJ14466),ORAI2 (C7prf19), and ORAI3 (MGC13024). See Peel et al., Am J Respir CellMol Biol. 2008 June; 38(6): 744-749 (published online Jan. 31, 2008),which is incorporated herein by reference.

One of the characteristics of Drosophila mutant itpr cells is thatintracellular calcium release is compromised when the cells are exposedto a stimulant. See, for instance, FIG. 6( a). Provided below are moredetails about a specific itpr mutant gene, known as “itpr-ku,” whichexhibits compromised calcium ion release characteristics, but thepresent technology is not limited to only this particular itpr mutation.

Another characteristic of the mutant itpr Drosophila cell is that normalcalcium ion release can be restored upon the expression of a dOraigenes. This is because the genes encoding the SOC channel (Orai1) andthe store Ca2+ sensor (Stim1) are known to maintain intracellular Ca2+store levels ([Ca2+]ER) in stimulated T-cells, especially since thereplenishment of [Ca2+]ER in T-cells is required for their prolongedactivation. See Feske, S. et al., Nat Immunol 2, 316-324 (2001).Homologs of mammalian Orai and Stim exist in Drosophila as single genesand perform similar cellular functions in S2 cells, derived from aprimary culture of late stage Drosophila embryos, where their depletionby gene specific double-stranded RNA (dsRNA) leads to abrogation ofstore-operated Ca2+ entry (SOCE). See Feske, S. et al., Nature, 441,179-185 (2006); Vig, M., et al., Science, 312, 1220-1223 (2006); Zhang,S. L. et al., Proc Natl Acad Sci USA, 103, 9357-9362 (2006), which areall incorporated herein by reference. Hence, the presence of dOrai2 inan itprku background restores stimulated Ca²⁺ release to wild typelevels.

There are several characteristics of mutant itpr cells that provide fora useful model system including, but not limited to, the cells have acompromised calcium ion release upon stimulation, and the abnormal ornon-wild-type calcium store and compromised calcium releasecharacteristics are restored upon expression of an Orai gene.

The present technology is not limited to the use of Drosophila cells.Any cell type that exhibits these characteristics may be used. Suchcells include, but are not limited to, any mammalian, insect, reptile,fish, or bird cell that has a mutant itpr gene, or a mutant homolog of aDrosophila itpr gene, that has the same or similar compromised calciumion release characteristics as those of, for example, an itpr-kuDrosophila mutant cell. A mammalian cell includes but is not limited toa human cell, a primate cell, a mouse cell, a rat cell, a chicken cell,a cattle cell, a sheep cell, a hamster cell, a rodent cell, a pig cell,a cat cell, or a dog cell. A cell may have any tissue origin. That is acell may be obtained from neuronal tissue, brain tissue, liver tissue,kidney tissue, cardiac tissue, pancreatic tissue, stomach tissue,lymphatic tissue, retinal tissue, intestinal tissue, or reproductiveorgan tissues, or any tissue or organ from a body.

Such cells can be isolated in several different ways. See, for instance,the series of volumes entitled “Human Cell Culture” by Koller, andPalsson (Eds). For instance, cells can be purified from blood;mononuclear cells can be enzymatically released from soft tissues (forexample by applying collagenase, trypsin, or pronase) to break down theextracellular matrix and thereby release the cells; or via explantculture where tissue can be submerged in growth media and the resultantcells that are grown isolated from that medium. Thus, cells can beisolated from tissue suspension, grown in a culture dish, cultured inserum-free or chemically defined media, fused with other cells toproduce hybrid cells, such as a hybridoma. See Bruce Alberts, et al.,MOLECULAR BIOLOGY OF THE CELL (2002), which is incorporated herein inits entirety. There are also two other protocols for primary cellculture. One is for temporary cell culture studies (up to a few days)which is described in Banerjee et al., J Neurosci. 26(32):8278-88(2006), which is incorporated herein by reference, and also at page 126of DROSOPHILA CELL IN CULTURE by Guy Echalier, Academic Press (1997),which is also incorporated herein by reference. For obtaining permanentcell lines the procedure is different and is described in Chapter 3(“Drosophila continuous cell lines”) of Echalier.

Cells that are obtained directly from an organism or individual areprimary cells, which are able to replicate and divide for a period oftime before they senesce. By contrast, an immortalized cell line canproliferate indefinitely; most vertebrate cells undergo senescence.Human somatic cells do not permanently express telomerase, whichnormally maintains the telomeres that otherwise shorten every celldivision. Human fibroblasts, however, can be coaxed to proliferateindefinitely by providing them with the gene that encodes the catalyticsubunit of telomerase; they can then be propagated as an “immortalized”cell line. See Alberts (supra) at Vol. III, Chapter 8 (ManipulatingProteins, DNA and RNA). Some human cells, however, still stop dividingeven in the presence of telomerase because the culture conditionsinadvertently arrest the cell cycle. Thus, these cellular arrestingmechanisms have to be switched off, which can be accomplished byintroducing certain cancer-promoting oncogenes derived from tumorviruses. Unlike human cells, however, most rodent cells do not turn offtelomerase and therefore their telomeres do not shorten with each celldivision. In addition, rodent cells can undergo genetic changes inculture that inactivate their checkpoint mechanisms, therebyspontaneously producing immortalized cell lines. Having said that, humanembryonic stem cell lines, obtained from the inner cell mass of theearly embryo, can proliferate indefinitely while retaining the abilityto give rise to any part of the body.

Although all the cells in a cell line are very similar, they are oftennot identical. The genetic uniformity of a cell line can be improved bycell cloning, in which a single cell is isolated and allowed toproliferate to form a large colony. In such a colony, or clone, all thecells are descendants of a single ancestor cell. One of the mostimportant uses of cell cloning has been the isolation of mutant celllines with defects in specific genes. Studying cells that are defectivein a specific protein often reveals valuable information about thefunction of that protein in normal cells.

Once isolated, cells can be grown and maintained at an appropriatetemperature and gas mixture (typically, 37° C., 5% CO₂ for mammaliancells) in a cell incubator, although precise growth conditions vary eachcell type, and variation of conditions for a particular cell type canresult in different phenotypes being expressed. Similarly, recipes forgrowth media can vary, too, and include, but are not limited to variableingredients and components, such as pH, glucose concentration, growthfactors, and the presence of other nutrients. The growth factors used tosupplement media are often derived from animal blood, such as calfserum.

Cells can be grown in suspension or adherent cultures. Some cellsnaturally live in suspension or can also be modified to survive insuspension cultures so that they can be grown to a higher density thanadherent conditions would allow. By contrast, most cells obtained fromsolid tissues are adherent cells and require a surface to grow anddifferentiate. One type of adherent culture is organotypic culture whichinvolves growing cells in a three-dimensional environment as opposed totwo-dimensional culture dishes. This culture system is biochemically andphysiologically more similar to in vivo tissue. In the case of adherentcultures, the media can be removed directly by aspiration and replaced.

Once cells have been isolated and grown in culture, they can besubsequently subcultured by a process called “passaging.” This involvesregularly transferring a small number of cells into a new vessel. Cellscan be cultured for a longer time if they are split regularly, as itavoids the senescence associated with prolonged high cell density.Suspension cultures are easily passaged with a small amount of culturecontaining a few cells diluted in a larger volume of fresh media. Foradherent cultures, cells first need to be detached; this is commonlydone with a mixture of trypsin-EDTA, however other enzyme mixes are nowavailable for this purpose. A small number of detached cells can then beused to seed a new culture.

Common immortalized human cell lines include but are not limited to 3T3fibroblast (mouse), BHK21 fibroblast (Syrian hamster), MDCK epithelialcell (dog), HeLa epithelial cell (human), PtK1 epithelial cell (ratkangaroo), L6 myoblast (rat), PC12 chromaffin cell (rat), SP2 plasmacell (mouse), COS kidney (monkey), 293 kidney (human); transformed withadenovirus, CHO ovary (chinese hamster), DT40 lymphoma cell forefficient targeted recombination (chick), R1 embryonic stem cells(mouse), E14.1 embryonic stem cells (mouse), H1, H9 embryonic stem cells(human), S2 macrophage-like cells (Drosophila), and BY2 undifferentiatedmeristematic cells (tobacco). See Table 8.2 (Some Commonly Used CellLines) of Molecular Biology of the Cell, III, Chapter 8, by Alberts(4^(th) Ed.) (2002).

Any of these cells, primary cell cultures, or immortalized cell lines,may be further modified to express a Drosophila mutated itpr gene or thehomolog of the itpr gene that is equivalent to itpr that is endogenousto the genome of a particular cell from another species. An itpr mutantgene of the present technology is itpr^(ku), which is an abbreviation ofthe genotypic representation of the underlying heteroallelic mutantcombination itpr^(ka1091/ug3). See Joshi et al., Genetics 166, 225-236(2004), which is incorporated herein by reference in its entirety. Themutated residue in itprka1091 (Gly to Ser at 1891) lies in themodulatory domain while in itprug3 it lies in the ligand binding domainat position 224 (Ser to Phe); both residues are conserved in mammalianInsP3R isoforms. See Srikanth, S. et al., Biophys J, 86, 3634-3646(2004). Three genes encode for a family of InsP3Rs in mammalian cells,including humans, and other vertebrates. The three full-length aminoacid sequences are 60-80% homologous overall, with regions, includingthe ligand-binding and pore domains (discussed below), having muchhigher homology. See Foksett et al. (supra), subsection B “InsP3RDiversity.” Invertebrates appear to express only a single InsP3R, mostclosely related to the type 1 isoform. In mammals, the InsP3R isubiquitously expressed, perhaps in all cell types. The three channelisoforms have distinct and overlapping patterns of expression, with mostcells outside the central nervous system expressing more than one type.The itpr-equivalent mutant residues could directly affect InsP3Rinteractions with a store Ca²⁺ regulating molecule like STIM. See Luik,R. M. et al., Nature, 454, 538-542 (2008); Taylor, C. W., Trends BiochemSci, 31, 597-601 (2006). Measurement of InsP3-mediated Ca²⁺-release frommicrosomal vesicles of itpr^(ku) shows a significant reduction ascompared to wild-type, non-itpr^(ku) cells. See Srikanth et al., BiophysJ, 86, 3634-3646 (2004). The ability of itpr^(ku) to maintain elevated[Ca²⁺]ER at 25° C. suggests a possible interaction between thisheteroallelic combination and Orai/STIM. See also Redondo, P. C., etal., Biochim Biophys Acta 1783, 1163-1176 (2008).

To produce transgenic fruit flies, the mutated itpr gene fragment isinserted between the two terminal sequences of a Drosophila transposon,the P element. The terminal sequences enable the P element to integrateinto Drosophila chromosomes when the P element transposase enzyme isalso present. To make transgenic fruit flies, this P element, containingthe mutated sequence, is injected into a Drosophila embryo along with aseparate plasmid containing the gene encoding the transposase. When thisis done, the injected gene often enters the germ line in a single copyas the result of a transposition event. An example of such mutant cellsare Drosophila larval primary neurons, which can be obtained fromstandard mechanical and enzymatic methods of dissociation of 3^(rd)instar larval brains. See Producing Primary Cultures of DrosophilaLarval Neurons in the Overview of Examples below for specificmethodological details. A human cell also may be made into a primarycell culture or immortalized in a human cell line as described herein.

Mammalian versions of such mutated Drosophila genes and transgenic fruitflies can also be made and used in the screening assay described here.For instance, mammalian InsP3R gene isoforms, InsP3R-1, InsP3R-2, andInsP3R-3, can be mutated in the same way as the Drosophila itprka1091and itprug3 mutant genes, so as to effectively produce a mammalian cellthat exhibits compromised calcium ion release upon stimulation, like theitpr-ku mutant Drosophila cell (which contains the −1091 and −ug3mutations in the Drosophila itpr gene). These inositol1,4,5-trisphosphate receptors (InsP3Rs) are a family of Ca²⁺ releasechannels localized predominately in the endoplasmic reticulum of allcell types. They function to release Ca²⁺ into the cytoplasm in responseto InsP₃ produced by diverse stimuli, generating complex local andglobal Ca²⁺ signals that regulate numerous cell physiological processesranging from gene transcription to secretion to learning and memory. SeeFoskett et al., Physiol. Rev. 87: 593-658, (2007), which is incorporatedherein by reference. The InsP3R is a calcium-selective cation channelwhose gating is regulated not only by InsP₃, but by other ligands aswell, i such as cytoplasmic Ca²⁺.

Phospholipase C hydrolyzes membrane lipids to produce inositol1,4,5-trisphosphate (InsP₃), which diffuses in the cytoplasm and bindsto the InsP3R receptor, which is an intracellular ligand-gated Ca²⁺release channel localized primarily in the endoplasmic reticulummembrane. See Foskett et al. (“Introduction”). The ER is the major Ca²⁺storage organelle in most cells. ER membrane Ca²⁺-ATPases accumulateCa²⁺ in the ER lumen to quite high levels. Because the lumen containshigh concentrations of Ca²⁺ binding proteins, the total amount of Ca²⁺in the lumen may be >1 mM; the concentration of free Ca²⁺ has beenestimated to be between 100 and 700 μM. In contrast, the concentrationof Ca²⁺ in the cytoplasm of unstimulated cells is between 50 and 100 nM,3-4 orders of magnitude lower than in the ER lumen. This lowconcentration is maintained by Ca²⁺ pumps and other Ca²⁺ transporterslocated in the ER, as well as plasma, membranes. Upon binding InsP₃, theInsP3R is gated open, providing a pathway for Ca²⁺ to diffuse down thiselectrochemical gradient from the ER lumen to cytoplasm. Ca²⁺ in thecytoplasm moves by passive diffusion, at a rate that is reduced bymobile and immobile Ca²⁺ binding proteins acting as buffers.Consequently, microdomains with steep Ca²⁺ concentration gradients canrapidly form and dissipate near the mouth of an InsP3R Ca²⁺ channel. TheCa²⁺ concentration adjacent to the open channel may be 100 μM or more,whereas concentrations as close as 1-2 μm from the channel pore may bebelow 1 μM.

According to the present methodology, one embodiment is to disrupt allthree mammalian InsP3R isoforms so that there is no functionalendogenous inositol 1,4,5-trisphosphate receptor expressed in the cell.A cell culture can then be made from this triple knockout cell and thentransformed with a mutant version of any of the InsP3R genes, so thatthe cell expresses only the mutated version of one InsP3R isoform.Accordingly, one could use a DT-40 cell line where all three InsP3Rshave been knocked out or silenced according to standard techniquesdescribed herein, such as by RNAi, site-directed mutagenesis, orhomologous recombination. See also Kuhn & Wurst, GENE KNOCKOUT PROTOCOLSin the series entitled METHODS IN MOLECULAR BIOLOGY, Vol. 530 (2^(nd)Ed., 2009, Humana Press) XVI, which is incorporated herein by reference.See also Barnett & Kontgen, Gene Targeting in a Centralized Facility atpage 65 of Gene Knockout Protocols, Vol. 158 of METHODS IN MOLECULARBIOLOGY by Tymms and Kola (2001, Humana Press), which is incorporatedherein by reference. For a review on the use of RNA interference toknockdown or silence genes see Voorhoeve & Agami, Knockdown Stands Up,Trends Biotechnol., 21(1):2-4 (2003), which is incorporated herein byreference, and Xia et al., Transgenic RNAi: accelerating and expandingreverse genetics in mammals, Transgenic Research, 15:271-275 (2006),which is incorporated herein by reference. See also Hasan, G.,Biological Implications of Inositol 1,4,5-triphosphate signaling fromgenetic studies in multicellular organisms, Proc. Indian natn Sci Acad.B69, No. 5: 741-752 (2003), which is incorporated herein by reference,and which describes the role of InsP3R receptors and mutated InsP3Rgenes in Drosophila, C. elegans, and mice.

As mentioned, one particular type of cell that can be engineered so asto have knocked-out InsP3R genes is the DT40 cell line, which is achicken B cell line that permits efficient gene knockout targeting dueto its high homologous recombination activities. See Buerstedde et al.,The DT40 web site: sampling and connecting the genes of a B cell line,Nucleic Acids Research, Vol. 30, No. 1:230-231 (2002), which isincorporated herein by reference. A DT-40 cell line can be targetedaccording to standard methods described herein to knockout or silencethe three InsP3R gene isoforms. See also Method 19 Targeted Transfectionof DT40 Cells beginning at page 419 of REVIEWS AND PROTOCOLS IN DT40RESEARCH by Buerstedde & Takeda (2006) published by SpringerNetherlands. The triple mutated DT40 cell can then be engineered toexpress a mutated InsP3R gene, which can be, for example, mutated tocontain the equivalent point mutations of the Drosophila itpr-ku mutatedreceptor gene. Such a DT40 cell then can be used, according to the assayprotocols and methods presently described herein, to observe changes inintracellular calcium, and to monitor and record the effect(s) of acompound in modulating that intracellular calcium level. A cell that istherefore useful for the present assay is a human cell engineered tohave knocked out versions of all three InsP3R endogenous isoforms butwhich has been transformed with at least one of a mutated InsP3Risoforms that has been altered in the same, or equivalent, way as theDrosophila itpr-ku gene, and reintroduced and expressed in thattriple-knocked out human cell. Alternatively, two endogenous InsP3Rgenes may be knocked out according to standard techniques and the thirdendogenous InsP3R isoform mutated by site-directed mutagenesis orhomologous recombination with the desired itpr-ku-like mutations, whichwould avoid the need to retransform the cell with an expression cassettecomprising an exogenous mutated InsP3R sequence.

As mentioned, there are different ways to create a mutated gene. Oneway, a mutated itpr homolog gene can be introduced into cultured EScells via homologous recombination. Cells containing the introducedmutant allele can then be identified and cultured to produce manydescendants, each of which carries an altered gene in place of one ofits two normal corresponding genes. These altered ES cells can then beinjected into an embryo, such as a mouse embryo, whereafter the cellsbecome incorporated into the growing embryo. The resultant mouse that isborn will contain some somatic cells and some germ-line cells that carrythe mutated itpr/homolog gene. Accordingly, upon breeding, some progenywill contain the altered gene in all of their cells. These cells thencan be obtained and isolated and cultured according to the methodsdescribed above. These progeny, if mice, and if the mutation completelydeactivates the itpr/homolog gene are known as knockout mice.Accordingly, cells from knockout mice also can be isolated and culturedaccording to the techniques described herein for use in the presentassay.

Alternatively, site-directed mutagenesis can be used to target mutationsinto the itpr/homolog gene, such as the point mutations described abovefor itpr-ku mutations. A synthetic DNA oligonucleotide designed tocontain those mutations is then hybridized with single-stranded plasmidDNA that contains the version of the itpr gene to be altered. Theoligonucleotide serves as a primer for DNA synthesis by DNA polymerase,thereby generating a duplex that incorporates the altered sequence intoone of its two strands. After transfection, plasmids that carry thefully modified gene sequence are obtained. The appropriate DNA is theninserted into an expression vector so that the redesigned protein can beproduced in the appropriate type of cells, isolated as described above,for use in the presently-described screening assay. For additionalinformation on molecular biology cloning methods and site-directedmutagenesis, see Sambrook et al., MOLECULAR CLONING: A LABORATORYMANUAL, (2d Ed), published by Cold Spring Harbor Laboratory (1989),which is incorporated herein by reference, particularly Chapter 15 onSite-Directed Mutagenesis.

Foreign DNA can also be readily integrated into random positions of manyanimal genomes. In mammals, for instance, linear DNAs that areintroduced into mammalian cells are rapidly ligated end-to-end byintracellular enzymes to form long tandem arrays, which usually becomeintegrated into a chromosome at an apparently random site. If themodified chromosome is present in the germ line cells (eggs or sperm),the mouse will pass these foreign genes on to its progeny, as describedin the preceding section.

The screening assay of the present technology identifies those compoundsthat modulate, i.e., cause the increase or decrease of intracellularcalcium levels, of the itpr cell mutant. An aliquot of cells, forinstance those that have been obtained and isolated as described above,can be put into a new vessel, or into a well of a standard 96-wellplate, and the calcium imaged. Calcium ion imaging of the cells can beaccomplished by recording fluorescent intensity values before and afterstimulation of cells with candidate compounds. Fluorescence is measuredin arbitrary units (AU). For example, for a wild type cell, if theresting fluorescence is 600 AU, stimulation with 10 μM thapsigarginwould increase the pixel intensity value to 2500. This difference influorescent intensity is then normalized to the resting fluorescentintensity to determine ΔF/F (1300/600 which would be 3.167) the range ofwhich is plotted as a box plot. This calculation has been done for everycalcium imaging experiment where store calcium, SOCE and stimulatedrelease through InsP3R is measured. For measuring resting cytosolic Ca²⁺in primary Drosophila neurons of different genotypes, the ratiometricfluorescent dye Indo-1AM can be used. Accordingly, the resting calciumlevels and characteristics of an aliquot of cells can be obtained andrecorded. Then, a candidate compound can be added to the same or adifferent aliquot of the same cells and the calcium levels and patternsimaged again using the same technique and then compared to thenon-compound-added cells to determine how, if at all, the compoundchanged the intracellular calcium stores and release of calcium ions viathe itpr-ku mutant.

Such a screening assay can be performed on other cells from otherspecies which have been isolated and cultured, or immortalized,according to the techniques described herein and known to the skilledperson. A mammalian cell, for instance, can be used in a screening assayto determine if a candidate compound modulates the calcium store andcalcium pattern of the mammalian cell. Thus, before so using a mammaliancell, a resting image of the mammalian cell which contains anequivalently-mutated itpr homolog gene, i.e., equivalent to theDrosophila itpr-ku mutant gene, can be taken and recorded. As apreliminary matter, one could compare the mutated mammalian cell'scalcium image to that of the Drosophila itpr-ku mutant cell calciumpattern to determine any similarities or differences. Then, a candidatecompound can be added to the mammalian mutant cell and the mammalianmutant cell then re-imaged to obtain a post-administration calciumimage. By comparing the two images, before and after administration ofthe compound, it can be determined what effect, if any, the compound hason the calcium store and flux of the mammalian mutant cell.

The screening assay does not have to be comprised of only two timepoints (before and after) of administration of the compound. Multiple“doses” of a compound can be administered to an aliquot of cells overtime, which are sequentially imaged for changes in calcium ion storageand flux; or a compound can be added to separate individual aliquots ofcells over a period of time and separate images obtained for eachaliquot so that any changes in calcium imaging can be followed over thatperiod of time.

In one embodiment, therefore, a compound that fully or partiallyrestores the intracellular calcium levels of the itpr cell mutant to alevel that is at, or which approximates, the intracellular calcium levelof a non-mutant cell, is a compound that can be used to modulateintracellular calcium levels. Thus, a compound that modulates calciumlevels is useful for treating diseases characterized by abnormalstore-operated, intracellular calcium levels.

Accordingly, a method of the present technology provides for theidentification of a compound that modulates store-operated calcium entrylevels in a cell, comprising providing a test compound to an itpr mutantcell; and determining whether the test compound increases or decreasesthe abnormal store-operated, intracellular calcium level of the itprmutant cell.

The compound can be added directly to such cultures of itpr mutant cellsor to an itpr mutant cell line or to cells obtained from an organism,such as from a mammal, e.g., human cells, Drosophila fruit flies, thathave been isolated and/or cultured from the organism. The concentrationof the test compound to be used is determined empirically so as toobtain a measurable fluorescence difference. In one embodiment, thecompound modulates the calcium level of the itpr mutant cell such thatthat level changes in the direction toward approximating the calciumlevel of a control cell. That is, the mutant cell calcium level may beelevated compared to a control cell in which case the compound, when itis provided to the cell, lowers the elevated calcium level so that thecalcium level moves in the direction toward the calcium level of acontrol cell.

Conversely, the mutant cell calcium level may be depleted compared to acontrol cell in which case the compound, when it is provided to thecell, increases the elevated calcium level so that the calcium levelmoves in the direction toward the calcium level of a control cell. Acompound that increases or decreases the calcium release through InsP3Rand/or changes the store operated calcium entry in an itpr-ku mutantcell is therefore a compound that modulates store-operated calcium entrylevels. The level of calcium may be monitored before, during, or afteradministration of the compound, such as at discrete endpoints or atconstant intervals, such as every 5 minutes over the course of a setperiod of time. The calcium levels also can be compared to the calciumlevel of a control cell. A control cell may be, for instance, (i) awild-type, normal cell, (ii) a cell with a dOrai/Kum⁻¹⁷⁰; itpr^(ku)mutant genotype, or (iii) a cell without the itpr^(ku) mutant genotype.An example of a control cell is a cell from the brain of a wild-typeCanton-S Drosophila strain. Accordingly, a test compound that increasesor decreases the calcium level in an itpr mutant cell, such as in theitpr^(ku) mutant cell described herein, or in a cell from a differentspecies which contains an equivalently mutated itpr/InsP3R gene is adesirable compound that modulates store-operated calcium entry levels.

“Modulates” is commonly understood to reflect a variation in aparticular parameter. Thus, as used herein, modulation refers to thechange in level of intracellular calcium ions before and after acompound has been provided to a mutant cell. That is, the effect of acompound on a cell of the present technology may be to increase ordecrease the level of intracellular ionic calcium in that cell. Thedegree to which the intracellular calcium level changes so as it closerapproximate the intracellular ionic calcium level of that of anon-mutant, i.e., control, cell is indicative of the compound's abilityto fully or partially “restore” the abnormal intracellular ionic calciumlevel of the itpr mutant cell to that of a control cell. A control cellmay be, for instance, (i) a wild-type, normal cell, (ii) a cell with adOrai/Kum⁻¹⁷⁰; itpr^(ku) mutant genotype, or (iii) a cell without theitpr^(ku) mutant genotype. Quantitative comparative data concerninglevels of store-operated calcium between itpr-ku cells and “control”cells are show in FIG. 6( e). The modulation of calcium levels thereforeencompases either increases and decreases in calcium levels of themutant itpr cell, such as the mutant itpr^(ku) cell.

A mutant itpr cell that has been exposed to a modulatory compound maysubsequently have an intracellular ionic calcium level that is differentfrom the intracellular ionic calcium level of the same mutant itpr cellwhich has not been exposed to the modulatory compound. That is, themodulatory compound may increase the intracellular ionic calcium levelof the mutant itpr cell or decrease the intracellular ionic calciumlevel of the mutant itpr cell. Accordingly, after the modulatorycompound has been provided to the mutant itpr cell the intracellularionic calcium level of the mutant itpr cell may be near to that ofintracellular ionic calcium levels of a wild type cell or some suchcontrol cell. Accordingly, after the modulatory compound has beenprovided to the mutant itpr cell, the mutant itpr cell store-operatedintracellular ionic calcium level may be described in terms of whetherthat level is that of the wild type calcium level or in terms of apercentage of wild type ionic calcium levels. For instance, afterproviding a compound to an itpr mutant cell, the store-operatedintracellular ionic calcium level may increase to a level orconcentration that is half the concentration of the wild type ioniccalcium level, i.e., the itpr mutant cell has a post-compound exposureionic calcium that is about 50% that of the wild type ionic calciumlevel/concentration. Or the compound may effectuate an increase instore-operated ionic calcium levels such that the resultantconcentration of ionic calcium after the compound has been provided toit is almost identical to the wild type ionic calcium level, e.g.,80-99%, or 100% identical, to the ionic calcium level of the wild typecell measured under the same conditions. Accordingly, after providing acompound to an itpr mutant cell, the itpr mutant cell may have astore-operated intracellular ionic calcium level that is about 50% ofthat of a control cell intracellular calcium level, about 55% of that ofa control cell intracellular calcium level, about 60% of that of acontrol cell intracellular calcium level, about 65% of that of a controlcell intracellular calcium level, about 70% of that of a control cellintracellular calcium level, about 75% of that of a control cellintracellular calcium level, about 80% of that of a control cellintracellular calcium level, about 85% of that of a control cellintracellular calcium level, about 90% of that of a control cellintracellular calcium level, about 95% of that of a control cellintracellular calcium level, or about 100% of that of a control cellintracellular calcium level. Accordingly the level of intracellular,store-operated ionic calcium may approximate the corresponding level ina control cell after exposure to the compound. A control cell of thepresent technology may be (i) a wild-type, normal cell, (ii) a cell witha dOrai/Kum⁻¹⁷⁰; itpr mutant genotype, or (iii) a cell without the itprmutant genotype. Or the compound may effectuate a decrease instore-operated ionic calcium levels such that the resultantconcentration of ionic calcium after the compound has been provided toit is lowered and almost identical to the wild type ionic calcium level,e.g., 80-99%, or 100% identical, to the ionic calcium level of the wildtype cell measured under the same conditions. Accordingly, afterproviding a compound to an itpr mutant cell, the itpr mutant cell mayhave a store-operated intracellular ionic calcium level that becomesreduced until it is near or at the level of intracellular ionic calciumof a control cell. Taking the change as an absolute value, the itprmutant cell may have a store-operated intracellular ionic calcium levelthat is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about100% of that of a control cell intracellular calcium level. The itprmutant cell may have a store-operated intracellular ionic calcium levelthat is a range of about 50%-100%, 55%-100%, 60%-100%, 65%-100%,70%-100%, 75%-100%, 80%-100%, 85%-100%, 90%-100%, or about 95%-100% ofthat of a control cell intracellular calcium level.

Thus the present technology concerns changes toward similarity instore-operated calcium levels that result from the exposure of the itprmutant cell to a compound that increases that cell's calcium levels ordecreases that cell's calcium levels so that the resultant change istoward a level similar to that of a wild-type or control cell.

The determining whether a test compound modulates the abnormalstore-operated, intracellular ionic calcium level of the itpr^(ku) cellcan be achieved by performing calcium imaging of the store-operatedcalcium entry environment of the itpr^(ku) cell, and recording thecalcium imaging patterns before and after the addition of the testcompound to the itpr^(ku) cell, and comparing the calcium imagingpatterns of the itpr^(ku) cell with the calcium imaging of thestore-operated calcium entry environment of a control cell, wherein thechange toward similarity in calcium imaging patterns between the twocells indicates the test compound can modulate the store-operatedcalcium entry level of the itpr^(ku) cell.

Thus, according to the present technology, a compound modulates calciumlevels in the store-operated calcium entry environment if the calciumimaging pattern of the store-operated calcium entry environment changesin the direction of matching the calcium imaging pattern of (i) awild-type, normal cell, (ii) a cell with a dOrai/Kum⁻¹⁷⁰; itpr^(ku)mutant genotype, or (iii) a cell without the itpr^(ku) mutant genotype;or the calcium imaging pattern of the store-operated calcium entryenvironment matches or is equivalent to the calcium imaging pattern of(i) a wild-type, normal cell, (ii) a cell with a dOrai/Kum⁻¹⁷⁰;itpr^(ku) mutant genotype, or (iii) a cell without the itpr^(ku) mutantgenotype.

A compound of the present technology, which modulates store operatedcalcium levels, includes, but is not limited to, e.g., a small molecule,an inorganic compound, an organic compound, a biomolecule, a chemical, aprotein, a peptide, a lipid (or derivative thereof), a carbohydrate(derivative thereof), or a nucleic acid. Such compounds may affect anyaspect of the calcium store, such as opening and closing of the storeoperated calcium channel (SOC), in order to modulate the ionicconcentration of calcium within an itpr mutant cell of the presenttechnology. Thus, the present technology is not limited to anyparticular cellular, chemical, or genetic target with which the compoundinteracts or regulates in order to directly or indirectly modulate ioniccalcium levels.

With respect to a nucleic acid, a compound may be a gene that encodes aprotein, a DNA or RNA oligonucleotide, or a type of RNA, such assingle-stranded sense or antisense RNA, or an RNA duplex, such as ahairpin loop RNA or a duplex formed from two annealing single strandedRNA strands. Thus, the present technology contemplates the introductionof a nucleic acid compound into an itpr mutant cell that causes a changein expression of one or more genes in the itpr mutant cell (such as, butnot limited to, by sense or antisense suppression or RNA interference,RNAi), which in turn results in a change or modulation in calcium levelsin the itpr mutant cell. Such nucleic acids may target one or morestore-operated channel genes, and related genes, thereby downregulatingtheir expression. For example, transient receptor potential (TRP) genemutants are associated with defective calcium ion influx. A number ofmammalian homologs of TRP have been found, and the TRP superfamilyincludes more than 20 related cation channels. These TRP channels can beclassified into three major subfamilies: TRPC, TRPV, and TRPM. The TRPCsubfamily exhibits the greatest sequence homology to Drosophila TRP.TRPC1, for instance, is involved in store-operated entry, whileoverexpression of TRPC3 enhances store-operated calcium. See Parekh andPutney, Physiol. Rev. 85: 757-810, 2005, which is incorporated herein byreference in its entirety.

A variety of mechanisms are known to directly or indirectly affectcalcium entry through store-operated calcium channels, such as but notlimited to (i) rapid inactivation (such as using fast calcium chelatorslike BAPTA); (ii) store refilling (and effect of thapsigargin); (iii)slow inactivation (effects of EGTA and calmodulin); and the effects ofcertain other compounds, e.g., the effect of sphingomyelinase,sphingosine, and ceramides (all of which reduce thapsigargin-evoked Ca²⁺entry) as targets in the sphingomyelin pathway that may regulatestore-operated influx; the effect of nitric oxide (NO), via cGMP andthen cGMP-dependent protein kinase, on stimulating Ca²⁺ reuptake intothe stores; the effect of protein kinase C on store-operated influx ofcalcium; and the effect of arachinoic acid as an inhibitor ofstore-operated calcium entry. Other examples of modulatory compoundsinclude Loperamide, which is a common antidiarrheal agent (see Harper etal., Proc Natl Acad Sci USA., 94(26): 14912-14917 (1997), which isincorporated herein by reference in its entirety); and the pyrazolederivative, YM-58483. See Ishikawa et al., J. Immunol., 170: 4441-4449(2003), which is incorporated herein by reference in its entirety.

The central role of calcium influx pathway in so many physiologicalsystems makes the itpr mutant cell assay of the present technology auseful tool for identifying compounds that modulate and affectstore-operated calcium ion influx and storage levels. The present assayidentifies compounds useful for treating diseases where alteredintracellular Ca²⁺ signaling or homeostasis is or may be a causativeagent. Exemplary diseases in this regard include, but are not limited tospino-cerebellar ataxia (Banerjee, S. & Hasan, G., Bioessays 27,1035-1047 (2005)), which arises by heterozygosity of the IP3R1 gene (vande Leemput, J. et al., PLoS Genet, 3, e108 (2007)); severe combinedimmuno-deficiency due to a mutation in Orai1 (Feske, S. et al., Nature,441, 179-185 (2006); and Thompson, J. L. et al., J Biol. Chem.,284(11):6620-6 (2009)); and Darier's disease from a mutation in SERCA2(Sakuntabhai, A. et al., Nat Genet, 21, 271-277 (1999)).

There are also documented cases of immunodeficiencies apparently derivedfrom impaired store-operated entry, as well as evidence for a role ofstore-operated entry in acute pancreatitis. There also is growingevidence for a role for store-operated entry in the toxic effects ofenvironmental chemicals that affect Ca²⁺ homeostasis. Other categoriesof diseases which can benefit from modulators of store-operated calciumlevels includes, severe combined immunodeficiency, acute pancreatitis,Alzheimer's Disease, Toxicology (prolonged elevation of cytoplasmic Ca²⁺can be toxic to cells; a typical example of agents in this class isthapsigargin, mentioned above, which is capable of killing cells invitro by apoptosis, or acting as a tumor promoter in vivo). An exampleof a calcium ion-mobilizing environmental toxin is tributyltin, animportant component of marine paints which has been shown to accumulatein coastal waters and estuaries and in marine organisms, which activatesCa²⁺ entry. See Parekh and Putney, Physiol. Rev., 85: 757-810 (2005),which is incorporated herein by reference in its entirety.

Based on the underlying changes in intracellular Ca²⁺ properties in suchdiseases, the present technology provides a way to identify compoundswhich may fully or partially restore or otherwise modulate ionic calciumlevels in itpr^(ku) mutant cells. The compounds that are identified asable to modulate calcium levels in itpr^(ku) mutant cells are compoundsthat could be useful in modulating calcium levels in cells ofindividuals with such diseases. Thus, a modulatory compound of thepresent technology can be administered to an individual with a diseasecharacterized by an intracellular calcium abnormality, in such aneffective amount, and for a period of time, or under a particular dosageregime so as to help rectify that abnormality by modulating theintracellular ionic calcium level of cells of that individual.

EXAMPLES

The present technology is further illustrated by the following examples,which should not be construed as limiting in any way.

Overview of Examples

The Examples that follow below show that store-operated Ca²⁺ entrythrough the Orai/STIM pathway and the rate of clearance of cytoplasmicCa²⁺ by SERCA together shape intracellular Ca²⁺ response curves inDrosophila neurons. The development and function of the flight circuitappears most sensitive to these cellular Ca²⁺ dynamics, changes in whichhave a profound effect on its physiological and behavioral outputs.Other circuits such as those required for walking, climbing and jumpingremain unaffected.

The flow of information in a neural circuit goes through multiple stepswithin and between cells. Suppression experiments, such as the onesdescribed here, present a powerful genetic tool for understanding themechanisms underlying both the formation of such circuits and theirfunction. See Ganetzky, B. & Wu, C. F., Genetics, 100, 597-614 (1982).Out-spread wings, higher spontaneous firing and initiation of rhythmicfiring on air-puff delivery in itprku are suppressed by eitherincreasing the quanta (through introduction of hypermorphic alleles ofdOrai and by dOrai+ over-expression) or by increasing the perdurance(through mutant Kum170) of the intracellular Ca²⁺ signal (FIG. 7,central panels).

Flight ability and maintenance of flight patterns requires SOCE inaddition to increased quanta and perdurance of the Ca²⁺ signals,suggesting that SOCE in neurons contributes to recurring Ca²⁺ signalsessential for flight maintenance (FIG. 7, last panel).

The concerted activity of Ca²⁺ flux pathways is critical for both themorphology and activity of neuronal circuits 1. The results belowdemonstrate a requirement for Ca²⁺ influx through SOC channels inelectrically excitable cells, which very likely constitute the flightCPG. Aminergic, glutamatergic and insulin-producing neurons could assistin development and/or directly constitute the circuit. Biogenic amines,especially serotonin, have been reported to regulate axonal growth andalso control rhythmic behavior in other invertebrate systems. SeeBudnik, V. et al., J Neurosci, 9, 2866-2877 (1989); and Koert, C. E., etal., J Neurosci, 21, 5597-5606 (2001). The glutamatergic domain consistsof a wide range of inter-neurons and the motoneurons that directlyinnervate the indirect flight muscles that power wing beating duringflight. See Mahr, A. & Aberle, H., Gene Expr Patterns, 6, 299-309(2006). Insulin producing neurons on the other hand probably control thegrowth of functional neurons and synapses that establish connectivity inthe CPG circuit.

Producing Primary Cultures of Drosophila Larval Neurons

According to the present technology, primary cultures of Drosophilalarval neurons were plated in 200 μl Drosophila M1 medium (30 mM HEPES,150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, and 35 mM sucrose, pH7.2) supplemented with 10% fetal bovine serum (Invitrogen, USA), 50 U/mlpenicillin, 50 μg/ml streptomycin, and 10 μg/ml Amphotericin B asdescribed previously. See Wu et al., J. Neurosci. 1983 September;3(9):1888-99. Briefly, brain and the ventral ganglion complex weredissected from Drosophila 3^(rd) instar larvae of the appropriategenotypes. The brain tissue was mechanically dissociated using syringeneedles in Schneider's medium containing collagenase (0.75 μg/μl) anddispase (0.4 μg/μl) and incubated in the proteolytic medium for 20minutes to allow complete dissociation of the tissue. The lysatecontaining essentially single cells was then spun down, re-suspended inM1 medium (200 μl of M1 was used for lysates of four brains), and platedonto 35 mm culture dishes with a poly-lysine coated coverslip for thebottom. The cells were incubated at 22° C. for 14-16 h before imaging.

Example 1 Store-Operated Calcium Entry in Drosophila Neurons isDependent Upon Orai and STIM

Genes encoding the SOC channel (Orai1) and the store Ca²⁺ sensor (Stim1)are known to maintain intracellular Ca²⁺ store levels ([Ca2+]ER) instimulated T-cells. The replenishment of [Ca2+]ER in T-cells is requiredfor their prolonged activation. See Feske, S. et al., Nat Immunol 2,316-324 (2001). Homologs of mammalian Orai and Stim exist in Drosophilaas single genes and perform similar cellular functions in S2 cells,derived from a primary culture of late stage Drosophila embryos, wheretheir depletion by gene specific double-stranded RNA (dsRNA) leads toabrogation of store-operated Ca²⁺ entry (SOCE). See Feske, S. et al.,Nature, 441, 179-185 (2006); Vig, M., et al., Science, 312, 1220-1223(2006); Zhang, S. L. et al., Proc Natl Acad Sci USA, 103, 9357-9362(2006).

To investigate SOC channel activity in Drosophila neurons levels ofdOrai transcripts were reduced using double-stranded RNA (dsRNA) inprimary neuronal cultures derived from larval brains. SOCE was monitoredby Ca²⁺ imaging of cultured neurons in Ca²⁺ add-back experiments, afterdepletion of endoplasmic reticular (ER) stores with thapsigargin in verylow external Ca²⁺ (FIG. 1 a). SOCE was significantly reduced in neuronsexpressing dsRNA for dOrai (UASdOraiRNAi221 denoted as dsdOrai; FIG. 1b, c). Furthermore, the level of intracellular store Ca²⁺ ([Ca²⁺]ER) wassignificantly lower in these cells (FIG. 1 d) suggesting that Ca²⁺ entrythrough Drosophila Orai channels contributes to the maintenance of storeCa²⁺ in neurons.

To ascertain that the reduced SOCE observed in cells expressing dOraidsRNA is gene specific, SOCE was measured in two alternate conditions.Double stranded RNA for the ER Ca²⁺-sensor dSTIM, (UASdSTIMRNAi073denoted as dsdSTIM) and a ligand-gated extracellular Ca²⁺ channel,NMDAR1 (UASdNR1RNAi333 denoted as dsdNR1) were expressed in all neurons.Normal function of STIM is considered essential for Orai channelactivity, while SOCE is not predicted to change when levels of a plasmamembrane localized ligand-gated Ca²⁺-channel are reduced. Pan-neuralexpression of dsdStim followed by Ca²⁺ imaging revealed significantreduction of SOCE, [Ca²⁺]ER (FIG. 1 b, c) and resting cytosolic Ca²⁺([Ca²⁺]i; FIGS. 1 e and f). A significantly higher frequency of cellswith lower [Ca²⁺]i were present among the neuronal population withdsdSTIM. However, dsdOrai expression had no effect on [Ca²⁺]i suggestingdifferential efficacy of the two dsRNA strains for dOrai and dSTIM. Theefficacy of the dsRNA strains used in these experiments was confirmed bysemiquantitative RT-PCR.

Orai transcripts were consistently, reduced by pan-neural expression ofdOrai dsRNA (ElavC155GAL4/+; UASdOraiRNAi221; FIG. 8 a, b). There wassignificant reduction in levels of dSTIM transcripts (FIG. 8 c, d). Asexpected, reduction in the level of dNR1 transcripts did not affectstore Ca²⁺ or SOCE (FIGS. 8 e and f). These results demonstrate thatCa²⁺ influx, leading to replenishment of ER stores through the STIM-Oraipathway, is conserved in Drosophila neurons. Moreover, the single STIMencoding gene in Drosophila appears to regulate both [Ca²⁺]ER and[Ca²⁺]i. In mammalian systems these cellular properties are regulatedindependently by STIM1 and STIM2 respectively. See Brandman, O. et al.,Cell, 131, 1327-1339 (2007).

Example 2 Reduced SOCE in Drosophila Neurons Causes Flight Defects

In order to determine if reduced SOCE in Drosophila neurons affectsneuronal function, motor co-ordination defects were measured in theappropriate genotypes. No obvious changes were visible in larvaeexpressing dsRNA for either dOrai or dSTIM. The larvae were viable andpupated normally. However, adult flies with pan-neural expression ofdsdOrai and dsdSTIM had defective wing posture (FIG. 2 a, b). The wingsof these flies were held apart. They were examined for their free flightability by the “cylinder droptest” assay. See Benzer, S., Sci Am, 229,24-37 (1973).

Reducing Orai and STIM by dsRNA in neurons resulted in a significantloss of flight in adults. While greater than 50% flies with dOraiknock-down were flightless, dSTIM knock-down resulted in a complete lossof flight (FIG. 2 c). Expression of dsdOrai and dsdSTIM in glutamatergicneurons, which include the flight motor neurons, reduced flight abilityin ˜35% of adult Drosophila. The extent of flightless behavior wassignificant as compared with controls but less when compared withpanneural expression of dsdOrai and dsdSTIM, suggesting that therequirement for SOCE in flight is in flight motor neurons and otherneurons as well. Other adult motor activities such as walking andclimbing remained unaffected upon down-regulation of dOrai or dSTIM.

To understand how neuronal store Ca²⁺ and SOCE reduce flight ability,postsynaptic responses from the dorsal longitudinal indirect flightmuscles (DLMs) that power flight, were measured. Electrophysiologicalrecordings were obtained during tethered flight (initiated in responseto an air-puff stimulus) and at rest (FIG. 2 d, e, f). Non-fliers withpan-neural expression of dsdOrai and dsdSTIM, selected from the“cylinder drop” test were either unable to initiate rhythmic actionpotentials in response to an air puff stimulus or exhibited un-sustained(<5 s) and arrhythmic flight patterns (FIG. 2 d). Knock-down by dsdOraiand dsdSTIM in glutamatergic neurons lead to a milder change in flightpatterns as compared with pan-neural knock-down, consistent with a rolefor SOCE in non-glutamatergic inter-neurons in addition to theglutamatergic flight motor neurons. Recordings from resting DLMs ofthese flies revealed a high arrhythmic spontaneous firing rate of actionpotentials the frequency of which was significantly higher than WT (wildtype) or other control flies (FIG. 2 e, f).

Example 3 Over-Expression of dOrai+ in Neurons can Partially SuppressFlight Defects in Drosophila InsP3 Receptor Mutants

SOCE activation through Orai and STIM in vivo requires a signal fordepletion of intracellular Ca²⁺ stores. The effect of over-expressingdOrai in the genetic background of itpr mutants was tested. For thispurpose UASdOrai+ transgenic strains were generated and expressed inselected neuronal sub-domains. These include the glutamatergic domaintested above, the aminergic domain and the Drosophila insulin-likepeptide 2 producing neurons (Dilp2 neurons). Expression of two copies ofUASdOrai+ in Dilp2 neurons and the aminergic domain could partiallysuppress the altered wing posture of itprka1091/ug3 (hereafter referredto as itpr^(ku); FIG. 3 a). Though flight ability was not restored,there was initiation of flight patterns upon air puff delivery, normallycompletely lacking in itprku animals (FIG. 3 b). Moreover, spontaneoushyperactivity of the DLMs in itprku was suppressed to a significantextent by expressing UASdOrai+ either ubiquitously (hsGAL4-leaky orhsGAL4L at 25° C.; 13) or in the aminergic, Dilp2 and glutamatergicsub-neuronal domains (FIGS. 3 c, d).

To ascertain whether dOrai+ function is required during flight circuitformation in pupae and/or during acute flight in adults ubiquitousexpression of dOrai+ in UASdOrai+/hsGAL4L; itpr^(ku) organisms wasup-regulated by a heat-shock either in 24 hour pupae or in 1 day oldadults. In both conditions a significant number of flies could initiateflight in response to an air-puff. Thus the level of dOrai+ can modulateflight circuit activity both during its development and in adultfunction (FIG. 3 e). However, the flight patterns obtained were notsustained and appeared arrhythmic (FIG. 3 f) indicating that whiledOrai+ over-expression can suppress the flight defects and associatedphysiology of itpr mutant phenotypes to a significant extent, it isinsufficient to regain complete flight.

Example 4 Intracellular Calcium Homeostasis in Itprku is Restored byPan-Neuronal Expression of dOrai+

To understand the cellular basis of dOrai+ suppression of itpr mutantphenotypes, SOCE and [Ca²⁺]ER were measured in primary neurons fromitprku larval brains using Ca²⁺ addback experiments after maximaldepletion of stores by application of thapsigargin in very low externalCa²⁺ (FIG. 4 a, b). SOC influx was greatly diminished (FIG. 4 a, b)while [Ca²⁺]ER was significantly elevated in neurons derived from itprkularvae grown at 25° C., as compared to the WT. The mean [Ca²⁺]ER in itprmutant appeared twice as much as WT (FIG. 4 c). The percentage of cellswith detectable SOC was approximately 3-5% as compared to 70-80% in WT.

A change in dOrai or dSTIM transcript levels in itprku larvae was notobserved suggesting that a posttranscriptional change is responsible forthe altered cellular properties. Pan-neural overexpression of dOrai+ initprku neurons restored SOCE in itprku neurons to a significant extent.The percentage of cells with detectable SOCE increased to 70%. Moreover,[Ca²⁺]ER went back to WT levels. Over-expression of dOrai+ in WT neuronsdid not effect SOCE and [Ca²⁺]ER. The distribution of [Ca²⁺]i inneuronal cells derived from itprku was similar to WT (˜400 nM, FIG. 4 d,e). However, in neuronal cell populations derived from brains withpan-neuronal over-expression of dOrai+, [Ca²⁺]i was elevated (˜1 μM)both in WT and itprku backgrounds (FIG. 4 d, e).

The significance of deranged SOCE and [Ca²⁺]ER in itprku neurons wasdetermined by measuring these parameters in cells of itprku derived fromsecond instar larvae maintained at 17.5° C. itprku is a cold-sensitiveallelic combination and is lethal during the third instar larval stageat 17.5° C. See Joshi (2004) (supra).

SOCE and [Ca²⁺]ER in these conditions were similar to WT neurons grownunder identical conditions at 17.5° C. (FIG. 9 a, b). These data suggestthat itprku organisms up-regulate store Ca²⁺ at 25° C. as a compensatorymechanism to allow for survival at that temperature and that reducedSOCE may be a result of elevated store Ca2+. The observation that returnof [Ca²⁺]ER and SOCE to normal by dOrai+ over-expression, isinsufficient for restoration of complete flight in itprku suggests thatother aspects of intracellular Ca²⁺ signaling are essential for flightin these organisms.

Example 5 Restoration of Flight in Itprku by Dominant Alleles of dOraiand dSERCA

To investigate the additional properties of intracellular Ca²⁺ signalingrequired for flight, genetic interactions between itpr and dOrai werefurther probed. For this purpose mutant alleles with P-inserts in thedOrai gene were obtained. The two alleles obtained, and referred to asdOrai1 and dOrai2, both contain an EP{gy2} construct at a distance of 13bps from each other in the 5′ un-translated region (UTR) of the dOraigene (FIG. 10). See Bellen, H. J. et al., Genetics, 167, 761-781 (2004).The two dOrai alleles were initially tested for their interaction withitprku by measuring viability at 17.5° C.

Introduction of a single copy of either dOrai mutant allele couldsuppress cold-sensitive lethality of itprku (FIG. 11 a). A single copyof either dOrai allele also suppressed the wing posture defect of itprkugrown at 25° C. to a significant extent (FIG. 5 a) suggesting that bothdOrai1 and dOrai2 are gain of-function alleles (hypermorphs). Subsequentobservations support this conclusion further. The presence of a singlecopy of either dOrai allele in the background of itprku restored flightinitiation in response to an air-puff (FIG. 5 e) and suppressedhyperactivity of flight NMJs (FIG. 5 g, h). The extent of suppressionremained unaffected by introducing a second mutant allele of dOrai initpr mutant backgrounds, such as in the genotypes dOrai2/2; itprku anddOrai1/2; itprku.

The dOrai2/+ mutant allele can also partially suppress flight-relateddefects and reduced SOCE and [Ca²⁺]ER arising from pan-neuronalexpression of dsdSTIM (FIG. 5 d, f; FIG. 6 e, f). However, store Ca²⁺and SOCE in neurons heterozygous for dOrai2/+ is not significantlydifferent from WT (FIG. 11 b).

Flies of the genotype dOrai2/Kum¹⁷⁰; itpr^(ku) exhibited normal wings(FIG. 5 a) and normal levels of spontaneous electrical activity in DLMrecordings consistent with the previously demonstrated dominant effectof Kum170. See Luik, R. M et al., Nature, 454, 538-542 (2008).Strikingly, flight ability was restored in a significant number of thesetriple mutant flies (FIG. 5 b, c). This is in contrast to the completeloss of flight ability in itpr mutants and itpr, dOrai or itpr, dSERCAdouble mutant combinations. More than 60% of dOrai2/Kum¹⁷⁰; itpr^(ku)adults and nearly 50% of dOrai2/Kum170; itprku adults passed as “fliers”in the cylinder drop test assay (FIG. 5 b). Air puff delivery elicitedsustainable rhythmic flight patterns similar to wild-type in a highproportion of these flies (FIG. 5 c). Down-regulating SERCA functionthus restores or compensates for the additional intracellular Ca²⁺signaling deficits required for free flight, which are lacking in dOrai1or 2/+; itprku organisms.

Example 6 Ca²⁺ Release Through the InsP3 Receptor and SOCE TogetherContribute to Maintenance of Flight

Ca²⁺ release through the InsP3R was measured by stimulating neuronsectopically expressing the Drosophila muscarinic acetylcholine receptor(mAChR) with increasing concentrations of the agonist carbachol. SeeCordova, D. et al., Invert Neurosci, 5, 19-28 (2003); and Millar, N. S.et al., J Exp Biol, 198, 1843-1850 (1995). Pan-neuronal expression ofthe Drosophila mAChR had no measurable effect on viability or flight.For the WT InsP3R Ca²⁺ release increased as a function of carbacholconcentration (FIG. 9 d); it was greatly attenuated in itprku (FIGS. 6a, c, and FIG. 9 d). Expression of mAChR transcripts, as determined bysemiquantitative RT-PCR was similar in mutant and WT (FIG. 9 c). Ca²⁺release in larval neurons derived from itprku larvae grown at 17.5° C.was also significantly lower as compared with controls under similarconditions (FIG. 9 e).

Next, carbachol stimulated Ca²⁺ release in itpr^(ku) was measured in thepresence of dOrai2 and Kum170 double and triple mutant combinations.Kum⁷⁰ had no direct effect on Ca²⁺-release through the InsP3R uponcarbachol stimulation. The presence of dOrai2 in either dOrai2/+; itprkuor in dOrai2/Kum170; itprku organisms restored carbachol stimulated Ca²⁺release to wild-type levels (FIGS. 6 a, b). However this restoration isclearly not the only factor in flight maintenance since dOrai2/+; itprkuorganisms are flightless. Additional parameters were measured that arelikely to contribute to the flight rescue in triple mutants. Theseinclude perdurance of the carbachol stimulated Ca²⁺ peak, SOCE, [Ca²⁺]ERand [Ca²⁺]i.

The presence of a single copy of Kum170 delayed Ca²⁺ sequestrationfollowing carbachol stimulated release and led to greater perdurance ofthe Ca²⁺ peak; this effect of Kum170 was also present in cells derivedfrom dOrai2/Kum170; itprku organisms (FIG. 6 b, c). SOCE in neuronsderived from dOrai2/Kum170; itprku larvae, was significantly elevated ascompared to itprku and dOrai2/+; itprku and Kum170/+; itprku (FIG. 6 d,e). Thus, the combined effect of Orai2 and Kum170 on itprku is torestore near wild-type levels of InsP3 stimulated Ca²⁺-release, followedby a broader curve of Ca²⁺ persistence and normal SOCE. Consistent withthe known function of SERCA, Kum170 had a dominant effect and reducedlevels of store Ca²⁺ in all genotypes tested including Kum170/+; itprku(FIG. 6 f). Concurrent with the lower store, SOCE was greatly elevatedin Kum170 heterozygotes (FIG. 6 e). However, Kum170 was unable torestore SOCE in itprku neurons (FIG. 6 d, e). Interestingly, [Ca²⁺]ER indOrai2/+; itprku was also restored to normal, while cells withdetectable SOCE went up from 3-5% (in itprku) to 72%. Over all SOCEremained low suggesting that the significant effect of a single copy ofdOrai2 was restricted to a few critical neurons (FIG. 6 e). Importantly,in the triple mutants [Ca²⁺]ER remained low (FIG. 6 f), indicating thatsteady store Ca²⁺ levels do not effect flight directly but perhapscontribute to the higher SOCE observed. Larval neurons heterozygous fordOrai2 or Kum170/+ had elevated levels of basal cytosolic Ca²⁺ with orwithout itprku in the background (FIG. 11 c, d). Higher [Ca²⁺]i isunlikely to contribute directly to flight rescue since itpr mutants withhigh [Ca²⁺]i also exhibit flight defects.

Example 7 Drosophila Strains

The viable itpr heteroallelic combination used in this studyitprka1091/ug3 (referred to as itprku in the text) has single pointmutations in the itpr gene that were generated in an EMS (ethylmethanesulfonate) screen. Detailed molecular information on thesealleles has been published earlier. See Srikanth, S. et al., Biophys J,86, 3634-3646 (2004); and Joshi, R. et al., Genetics, 166, 225-236(2004). The UASmAChR transgenic strain on chromosome II was generated byinjecting embryos using a standard protocol with a pUASTmAChR constructgenerated from the Dm mAChR cDNA clone.

Ca-P60AKum170ts (referred to as Kum¹⁷⁰ throughout the text) was obtainedfrom Dr. K. S. Krishnan 23, dOrai11042 and dOrai20119 (referred asdOrai1 and dOrai2 respectively throughout the text) were procured fromthe Bloomington Stock Center (Bloomington, Ind.). UASRNAi strain fordOrai (12221), dSTIM (47073) and dNMDAR1 (dNR1; 37333), (referredthroughout the text as dsdOrai, dsdSTIM and dsdNR1 respectively) wereobtained from the Vienna Drosophila research centre (VDRC, Vienna). UASdOrai strains with and without CFP tags were generated by SuzanneZiegenhorn in the lab and mapped to chromosome 3.

The pan-neuronal GAL4 (used throughout the text) is ElavC155GAL4 mappedto X chromosome (Bloomington Stock Center, Bloomington, Ind.),glutamatergic GAL4 refers to OK371GAL4 36, aminergic refers to DdcGAL445, the GAL4 expressing in ILP2 producing neurons is Dilp2GAL4 46 andUbiquitous GAL4 refers to hsp70GAL4 (Heat shockLeaky) which has basalexpression at 25° C. Other fly strains were generated by standardgenetic methods using individual mutant and transgenic fly linesdescribed above.

Example 8 Larval Lethality and Staging

Staging experiments were done to obtain molting profiles ofheteroallelic mutant larvae as described previously (Joshi, supra).Timed and synchronized egg collections were done for 8 h at 25° C. Thecultures were then transferred to 17.5° C. at which temperaturedevelopment takes approximately double the time as compared todevelopment at 25° C. Heteroallelic and heterozygous larvae wereidentified using dominant markers (TM6Tb and CyoGFP), and separated at60 h AEL. They were transferred in 3 or more batches of 25 each toagarless cornmeal medium. Larvae were grown at 17.5° C. and screened atindicated time points for number of survivors and stage of development,determined by the morphology of the anterior spiracles. See Ashburner,M. Drosophila, a laboratory handbook. Cold Spring Harbor, N.Y.: ColdSpring Harbor Laboratory (1989).

Example 9 Heat Shock Experiments

Animals of the appropriate genotype were raised at 25° C. throughoutdevelopment. In the designated cases, a heat shock of 37° C. was givenfor 2 hrs to pupae 24 h after puparium formation (APF) or a 90 min heatshock at 37° C. was given to 1 day old adult flies.

Example 10 Flight Assay and Electrophysiology

Flight tests were performed as described by following minormodifications of the “cylinder drop assay” described previously. SeeBenzer, S., Sci Am 229, 24-37 (1973); Banerjee, S., Lee, J. et al., JNeurosci, 24, 7869-7878 (2004). Flies were tested in batches of 20 bydropping them into a 1 m long glass cylinder. Flies that fell throughdirectly into a chilled conical flask kept below were scored asnon-fliers and those that flew and stuck to the walls of the cylinderwere as scored fliers. The percentage of fliers was then determined.Computation of mean and standard error (SE) was performed using Origin7.5 software (MicroCal, Northampton, Mass.). Statistically significantdifferences between two groups were determined by two-way Student'st-tests for independent populations. Significant differences were takenat P<0.05. Physiological responses to an air-puff stimulus were recordedfrom the dorsal longitudinal muscles (DLMs) of the giant fiber pathway.Flies were anaesthetized briefly using diethyl ether and tethered usingnail polish to a thin metal wire inserted between the head and thethorax. Following recovery from anesthesia (˜4 h), an un-insulatedtungsten electrode sharpened by electrolysis was inserted into the DLM(fiber a), just beneath the cuticle.

A similar tungsten electrode was inserted into the abdomen served as thereference electrode. Flies were rested for 10 mins after insertion ofthe electrode before recording. Spontaneous firing was recorded byleaving the flies undisturbed in the dark for 2 minutes. Response to anair puff was then recorded from fiber a for 30 s by blowing a gentlepuff of air. All recordings were done using an ISO-DAM8A (WorldPrecision Instruments, Sarasota, Fla.) amplifier with filter set up of30 Hz (low pass) to 10 kHz (high pass). Gap free mode of pClamp8(Molecular Devices, Union City, Calif.) was used to digitize the data(10 kHz) on a Pentium 5 computer equipped with Digidata 1322A (MolecularDevices). Data were analyzed using Clampfit (Molecular Devices), andplotted using Origin 7.5 software.

Example 11 Primary Neuronal Cultures from Drosophila Larvae

Primary cultures of Drosophila larval neurons were plated in 200 μlDrosophila M1 medium (30 mM HEPES, 150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1mM CaCl₂, and 35 mM sucrose, pH 7.2) supplemented with 10% (v/v) fetalbovine serum (Invitrogen, USA), 50 U/ml penicillin, 50 μg/mlstreptomycin, and 10 μg/ml Amphotericin B as described previously. SeeWu et al., J. Neurosci., 3, 1888-1899 (1983). Briefly, brain and theventral ganglion complex were dissected from Drosophila 3rd instarlarvae of the appropriate genotypes. The brain tissue was mechanicallydissociated using syringe needles in Schneider's medium containingcollagenase (0.75 μg/μl) and dispase (0.4 μg/μl) and incubated in theproteolytic medium for 20 minutes to allow complete dissociation of thetissue. The lysate containing essentially single cells was then spundown, re-suspended in M1 medium (200 μl of M1 was used for lysates offour brains), and plated onto 35 mm culture dishes with a polylysinecoated coverslip for the bottom. The cells were incubated at 22° C. for14-16 h before imaging.

Example 12 Calcium Imaging in Larval Neurons

Larval neuron cultures were washed twice after growth for 14-16 h withDrosophila M1 medium and loaded in the dark with 2.5 μM Fluo-4 AM in M1medium containing 0.002% Pluronic F-127, for 30 minutes at roomtemperature. The fluorescent dyes were obtained from InvitrogenTechnologies, USA. After washing three times with M1, the cells werefinally covered with 100 μl of Ca²⁺ free M1 (CaCl₂ was substituted withequal concentration of MgCl₂) containing 0.5 mM EGTA and imaged within40 minutes of loading. For quantitative analysis, a field with severalcells was selected and imaged using the epifluorescence optics of aNikon TE2000 inverted wide field microscope with an oil objective (60×and 1.4 numerical aperture) lens. Fluo-4 was excited for 20 ms using 488nm wavelength illuminations from a mercury arc lamp. Emitted light wasdetected through a 505 nm bandpass filter (FITC filter set,41001-exciter HQ480/40, dichroic Q505LP, emitter HQ535/50; Chroma,Brattleboro, Vt.). For basal cytosolic Ca²⁺ measurements, cells wereloaded with 5 μM Indo-1 AM for 45 minutes at room temperature at the endof which the dye was removed, the cells washed and finally covered with100 μl of M1 containing 1 mM CaCl₂. Indo-1 in the cells was excited for300 ms using the Indo-1 filter set from Chroma (71002-exciter 365/10,dichroic 380 LP for emitter D405/30 and dichroic 440 LP with emitterD485/25).

Fluorescent images were acquired using the Evolution QExi CCD camera andIn vivo imaging software (Media Cybernetics, Silver Spring, Md.). Thetime lapse acquisition mode of the software was used to followfluorescence changes in the cells every 10 s or 15 s for 15 frames.Different concentrations of carbachol (Fluka, Mo., USA), thapsigargin(Invitrogen, USA) 10 μM or ionomycin (Calbiochem, SD) 10 μM were addedmanually approximately 15 s after the start of data acquisition. Formeasurement of store-operated Ca²⁺ entry, 1 mM CaCl₂ was added to thecells 225 s after thapsigargin addition. Images were acquired every 15s. As controls, a series of images were acquired with the same imagingprotocol without any additions. A total of 150-200 cells were analyzedfrom 5-7 dishes imaged for each genotype for each experiment.

Example 13 Data Analysis

For measuring fluorescence changes with time, images were processedusing ImagePro plus software, V1.33. Fluorescence intensity before(Fbasal′) and at various time points after addition of carbachol,thapsigargin, or CaCl₂ (Ft′) were determined. Background fluorescence(an area without any cells) was subtracted from the values of Ft′ andFbasal′ for each cell to obtain Ft and Fbasal The data were plottedusing Origin 6.0 software as follows: ΔF/F=(Ft-Fbasal)/Fbasal for everytime point. The maximum value of ΔF/F was obtained for every cell(Arrows in FIG. 1 b) and a box chart representing the data spread wasplotted. The rectangular boxes represent the spread of data pointsbetween 25-75% of cells, the horizontal line is the median and the smallsquare within represents the mean. Significant differences betweenmultiple groups of data were analyzed by one-way ANOVA orKolmogorov-Smirnov (K-S) test of significance as indicated in the figurelegends. For K-S test, cumulative frequency of cytosolic Ca²⁺ levels wasnormalized to the total number of cells analyzed for every genotype andplotted against the log of [Ca²⁺]i. The significance was calculatedbased on the maximum difference between the distributions referred to asthe K-S statistic. Significant differences were taken at P<0.05.

Measurement of resting cytosolic Ca²⁺ was done by obtaining values ofFbasal, Fmax (fluorescence at maximum saturation of the dye determinedby adding ionomycin to allow the cells to equilibrate to external Ca²⁺)and Fmin (fluorescence upon quenching all the free Ca²⁺) was determinedby adding 0.01% Triton-X and 1 mM EGTA). The values obtained weresubstituted in the Grynkiewicz equation:[Ca²⁺ ]i(μM)=(Fbasal−Fmin)/(Fmax−Fbasal)×Kd.

The published Kd value of 1.16 μM for Indo-1 in Drosophila S2 cells wasused. See Hardie et al., J. Neurosci., 16, 2924-2933 (1996).

Example 14 Reverse Transcription Polymerase Chain Reaction (RT-PCR)

RNA was extracted from fifteen individuals of the indicateddevelopmental stages using TRIZOL reagent (Invitrogen technologies,USA). Reverse transcription (RT) reactions were performed on 1 μg oftotal RNA using random hexaprimers (MBI, Fermentas) with Moloney murineleukemia virus (MMLV) reverse transcriptase (Invitrogen Technologies,USA) following standard protocols. 2 μl of the RT reaction product wasused to perform PCR under standard conditions using the followingprimers.

dOrai (SEQ ID NO. 1) Forward-5′ AGTTCTGCAGTGATCACCACTGG; (SEQ ID NO. 2)Reverse-3′ CCGCTACCCGTGGGACTGTTG. dSTIM (SEQ ID NO. 3) Forward-5′GAAGGCAATGGATGTGGTTCTG; (SEQ ID NO. 4) Reverse-3′ CCGAGTTCGATGAACTGAGAG.dNR1 (SEQ ID NO. 5) Forward-5′ AGGAGAAGGCCCTCAATCTC; (SEQ ID NO. 6)Reverse-3′ TAGTAGCAACGGAGCATGTG. rp49 (SEQ ID NO. 7) Forward-5′CCAAGGACTTCATCCGCCACC; (SEQ ID NO. 8) Reverse-3′ GCGGGTGCGCTTGTTCGATCC.dmAChR (SEQ ID NO. 9) Forward-5′ CAAGGACGAGTGCTACATCC; (SEQ ID NO. 10)Reverse-3′ CCTAAATCAGAAGGCTCCTCC.

Example 15 Calcium Imaging Assay

Pseudo-color images of store Ca²⁺ or [Ca²⁺]_(ER) and store-operated Ca²⁺entry (SOCE) in larval neurons of WT and itpr^(ku). Store Ca²⁺ wasmeasured by depleting stores upon addition of 10 μM thapsigargin (Tpg).SOCE was monitored by inclusion of Ca²⁺ (to a free concentration of 1mM) at t=225 s, to the extracellular buffer. Scale bar represents 10 μm.Warmer colors represent higher Ca²⁺. See FIG. 12( a). Single cell tracesof store-depletion and resulting SOCE by Ca²⁺ add-back experiments. SeeFIG. 12( b).

Example 16

One method of the present technology therefore is obtaining a humancell, such as a human fibroblast, or any human cell which expresses anInsP3R gene or ITPR (the homolog of the Drosophila itpr gene), which canbe used to create an immortalized human cell line by providing afunctional telomerase, after the human cell genome has been modified tohave a mutated InsP3R gene. See Alberts (supra) at Vol. III, Chapter 8(Manipulating Proteins, DNA and RNA). Site-directed mutagenesis can beused to target mutations into the fibroblast InsP3R gene itpr/homologgene, such as the point mutations described herein for itpr-kumutations. See Sambrook et al., Molecular cloning: a laboratory manual,(2d Ed), published by Cold Spring Harbor Laboratory (1989), which isincorporated herein by reference, particularly Chapter 15 onSite-Directed Mutagenesis. For an example of a Homo sapiens inositol1,4,5-triphosphate receptor, type 1 nucleotide sequence see NCBIReference Sequence: NM_(—)001099952.1, which is incorporated herein byreference.

Once the human itpr/InsP3R gene has been so mutated, it may be culturedinto a primary cell culture or an immortalized cell line according tothe methods described herein. Then, the calcium ion flux and storagecharacteristic of the mutated cell can be imaged and evaluated incomparison to a non-mutated, wild-type normal human cell of the sameorigin. A number of different images can be obtained such as byaccording to the assay of the preceding Example in order to obtain akind of “standard” resting calcium ion storage and flux traits of themutated human cells.

Next a candidate compound can be added to an aliquot of the human cellsand the cells then re-imaged after a period of time post-exposure to thecompound. The calcium image of the cells that have been exposed to thecompound can then be compared to the untreated human mutated cells andalso to the wild-type, non-mutated human cells, and a conclusion derivedas to whether the compound increases or decreases the intracellularcalcium ion level of the mutated human cell. That compound can then befurther evaluated for its ability to modulate calcium ion levels andthereby be a useful compound for, for example, a therapeutic fortreating a disease or disorder characterized by abnormal calcium levels.That is, after performing the calcium itpr/InsP3R cell screening assayof the present technology, the candidate compound can be used tomodulate the calcium level of a diseased cell type, or administered toan individual with an abnormal intracellular calcium ion storageproperties, so as to modulate that abnormal property and thereby reachor approximate normal calcium levels in that cell type or individual.

EQUIVALENTS

The present disclosure is not to be limited in terms of the particularembodiments described in this application. Many modifications andvariations can be made without departing from its spirit and scope, aswill be apparent to those skilled in the art. Functionally equivalentmethods and apparatuses within the scope of the disclosure, in additionto those enumerated herein, will be apparent to those skilled in the artfrom the foregoing descriptions. Such modifications and variations areintended to fall within the scope of the appended claims. The presentdisclosure is to be limited only by the terms of the appended claims,along with the full scope of equivalents to which such claims areentitled. It is to be understood that this disclosure is not limited toparticular methods, reagents, compounds compositions or biologicalsystems, which can, of course, vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the likeinclude the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 cells refers to groupshaving 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers togroups having 1, 2, 3, 4, or 5 cells, and so forth.

While various aspects and embodiments have been disclosed herein, otheraspects and embodiments will be apparent to those skilled in the art.The various aspects and embodiments disclosed herein are for purposes ofillustration and are not intended to be limiting, with the true scopeand spirit being indicated by the following claims.

What is claimed is:
 1. A kit for identifying a compound that modulatesstore-operated calcium entry (SOCE) level in a cell, comprising: atleast one mutant mammalian and/or avian cell, wherein the mutantmammalian and/or avian cell comprises a residual SOCE level andexpresses a single 1,4,5-inositol triphosphate receptor (InsP₃R) gene(itpr) product wherein the itpr gene encoding the single InsP₃R geneproduct consists of an itpr^(ka1091/ug3) mutant, and wherein theitpr^(ka1091/ug3) mutant contains G1891S and S224F point mutations; andat least one control cell.
 2. The kit of claim 1, wherein the controlcell is selected from the group consisting of: (i) a wild-type, normalcell, and (ii) a cell having wild-type calcium ion release.
 3. The kitof claim 1, wherein the mutant mammalian cell or the mutant avian cellis selected from the group consisting of a 3T3 cell, a BHK21 cell, anMDCK cell, a HeLa cell, a PtK1 cell, an L6 cell, a PC12 cell, an SP2cell, a COS cell a 293 cell, a CHO cell, a DT40 cell, an R1 cell, andE14.1 cell, an H1 cell, and an H9 cell.
 4. The kit of claim 1, furthercomprising a component for calcium imaging.
 5. The kit of claim 4,wherein the component for calcium imaging comprises a fluorescent dye.6. The kit of claim 5, wherein the fluorescent dye comprises fluo-4. 7.The kit of claim 1, further comprising thapsigargin.
 8. The kit of claim1, wherein the mutant mammalian cell is a murine cell.